CN109182562B

CN109182562B - miRNA apla-mir-25-42 related to follicular development of laying ducks as well as detection primer, inhibitor and application thereof

Info

Publication number: CN109182562B
Application number: CN201811421003.7A
Authority: CN
Inventors: 吴艳; 肖红卫; 张昊; 梁振华; 皮劲松; 潘爱銮; 蒲跃进; 孙静; 申杰; 杜金平; 白丁平
Original assignee: Institute of Animal Science and Veterinary of Hubei Academy of Agricultural Sciences
Current assignee: Institute of Animal Science and Veterinary of Hubei Academy of Agricultural Sciences
Priority date: 2018-11-27
Filing date: 2018-11-27
Publication date: 2021-05-04
Anticipated expiration: 2038-11-27
Also published as: CN109182562A

Abstract

The invention discloses miRNA apla-mir-25-42 related to follicular development of laying ducks as well as a detection primer, an inhibitor and application thereof, and belongs to the technical field of genetic engineering. The miRNA is apla-mir-25-42, and the nucleotide sequence of the miRNA is shown as SEQ ID NO. 1. The research of the invention shows that the miRNA can regulate follicular development by targeting the TGFB2 gene, reduce the expression of a cell proliferation marker gene CyclinB2 and promote the expression of an apoptosis marker gene BCL 2. The miRNA can be used as a marker for detecting the proliferation and differentiation conditions of the follicular granular cells of the laying ducks; the method makes up the existing vacancy in the field, provides theoretical basis and scientific basis for analyzing the genetic mechanism of follicular development of the laying ducks, and has important significance for researching the genetic essence of reproductive traits of the laying ducks, improving the reproductive capacity of the laying ducks and carrying out genetic auxiliary breeding.

Description

miRNA apla-mir-25-42 related to follicular development of laying ducks as well as detection primer, inhibitor and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to miRNA apla-mir-25-42 related to the follicular development of laying ducks, a detection primer, an inhibitor and application.

Background

The follicular development is closely related to the egg laying performance of the laying ducks, and directly influences the egg laying amount of the laying ducks. Research has shown that the follicular development of poultry is a very complex biological process, and people have already understood the follicular development pattern of poultry to some extent, but as an important factor for determining egg production, the specific regulation and control mechanism of follicular development still needs to be studied deeply.

However, miRNA markers for detecting follicular development status of laying ducks are currently lacking.

In view of this, the invention is particularly proposed.

Disclosure of Invention

To overcome the defects and shortcomings of the prior art, the present invention aims to provide a miRNA related to the follicular development of laying ducks. The miRNA can be used as a marker for detecting the proliferation and differentiation conditions of the follicular granular cells of the laying ducks and providing reference for judging the egg laying performance of the target laying ducks.

Another object of the present invention includes, but is not limited to, the use of the above-mentioned mirnas.

Another object of the present invention includes, but is not limited to, providing a detection primer for the above miRNA.

Another object of the present invention includes, but is not limited to, providing an inhibitor that inhibits the binding of the above-mentioned miRNA to its target gene.

Another object of the present invention includes, but is not limited to, providing a recombinant expression vector.

Another object of the present invention includes, but is not limited to, providing a method for regulating the development of follicles in laying ducks.

In order to achieve the purpose, the invention is realized by the following technical scheme:

with the coming of the genome era, the analysis of the overall characteristics of the expression mode of the gene related to the character on the whole genome level to reveal the molecular basis of the egg laying phenotype character formation of the laying ducks is one of the important research directions of the laying duck breeding work in the future. microRNA (miRNA) serving as a small single-stranded endogenous non-coding RNA can play a role in regulating and controlling the transcribed genes of the microRNA by degrading target mRNA or inhibiting translation. In recent years, numerous studies have shown that miRNAs play a key role in many physiological and pathological processes, such as cell differentiation and apoptosis, growth and development, glycolipid metabolism, inflammation, immune response, and tumorigenesis, among which some miRNAs that affect differentiation and proliferation of follicular granulosa cells have been screened and identified during follicular development.

The growth condition of the granulosa cells directly influences the development condition of the follicles, and the follicular granulosa cells can secrete gonadal hormones and growth factors to regulate the growth, differentiation and maturation of the follicular membrane cells and oocytes and accurately regulate the growth and development of the follicles. Therefore, the method has the advantages that the good follicular development is guaranteed, the regulation rule of follicular development is mastered, the reproductive capacity of the laying ducks is further improved, and the method has important significance for improving the production performance of the laying ducks.

However, no report is found on the research on the tissue-specific miRNA of the laying ducks, particularly the research on the follicle miRNA of the laying ducks and the regulation mechanism of the follicle miRNA. Therefore, the research level and the integrity of miRNA in the follicles of the laying ducks are urgently needed to be improved, so that a scientific theoretical basis is provided for the research on the genomes and non-coding RNAs of the follicles of the laying ducks, the control mechanism of the follicular development of the laying ducks is further understood, and a theoretical support is provided for improving the reproductive performance of the laying ducks.

According to the invention, two groups of follicle tissue samples at different development periods are subjected to high-throughput whole transcriptome sequencing, miRNA related to the development of the follicles of laying ducks is screened, and miRNA apla-mir-25-42 with high expression in white follicles is preliminarily selected through bioinformatics differential expression analysis, so that the expression of a cell proliferation marker gene CyclinB2 is promoted, and the expression of an apoptosis marker gene BCL2 is inhibited through the regulation of follicular development by targeting TGFB2 gene. The miRNA has important significance for researching the genetic nature of the reproductive traits of the laying ducks and improving the reproductive capacity of the laying ducks, and has important practical application value.

Based on the above, in the first aspect of the invention, the miRNA related to the follicular development of the laying duck is provided, wherein the miRNA is apla-mir-25-42, and the nucleotide sequence of the miRNA is shown as SEQ ID NO. 1.

In a second aspect of the invention, the application of the miRNA related to the follicular development of the laying duck as a molecular marker in detecting the proliferation and differentiation conditions of the follicular granular cells of the laying duck is provided.

Through research, the expression level of miRNA (namely the apla-mir-25-42 molecule) in the white follicles of the laying ducks is remarkably higher than that (P <0.01) in the yellow follicles of the laying ducks, and the association between the apla-mir-25-42 of the laying ducks and the follicular development of the laying ducks is found for the first time, so that the apla-mir-25-42 of the laying ducks can be used as a molecular marker for identifying the development condition of the follicles of the laying ducks, such as the proliferation and differentiation condition of follicular granulocytes, make up the existing vacancy in the field, and provide theoretical basis and scientific basis for analyzing the genetic mechanism of the follicular development of the laying ducks; in addition, the method has important significance for researching the genetic essence of the reproductive traits of the laying ducks, improving the reproductive capacity of the laying ducks and carrying out genetic auxiliary breeding.

In a third aspect of the invention, primers for detecting the above-mentioned mirnas are provided.

Further, in some embodiments of the present invention, the primers include an upstream primer shown in SEQ ID NO. 2, a downstream primer shown in SEQ ID NO. 3, and a LOOP primer shown in SEQ ID NO. 5.

In the fourth aspect of the invention, the inhibitor can inhibit the miRNA related to the follicular development of the laying duck from binding to a target gene, wherein the target gene is TGFB2 gene, and the binding site of the inhibitor and the miRNA is ATCCCA;

preferably, the inhibitor is selected from a segment of about 240bp in the 3' UTR of the TGFB2 gene.

Further, in some embodiments of the invention, the inhibitor sequence is set forth in SEQ ID No. 4;

preferably, restriction enzyme cleavage site sequences are added to the 5 'end and the 3' end of the inhibitor, respectively;

preferably, the restriction enzyme site sequence generates a sticky end upon cleavage;

more preferably, the restriction enzyme cutting site sequences are nucleotide sequences corresponding to a PmeI restriction enzyme cutting site and an XhoI restriction enzyme cutting site respectively;

more preferably, the PmeI cleavage site is located at the 5 'end and the XhoI cleavage site is located at the 3' end.

In a fifth aspect of the invention, there is provided a recombinant expression vector comprising an inhibitor as described above.

Further, in some embodiments of the invention, the marker gene in the recombinant expression vector is a dual-luciferase reporter gene;

preferably, the recombinant expression vector is obtained by linking the inhibitor to a reporter vector pmirGLO.

In a sixth aspect of the present invention, a kit for detecting the miRNA related to follicular development of laying ducks is provided, which contains a primer set for detecting the miRNA.

Further, in some embodiments of the invention, the primer set comprises a reverse transcription primer, an upstream primer and a downstream primer, wherein the sequence of the reverse transcription primer is shown as SEQ ID NO. 5; the sequence of the upstream primer is shown as SEQ ID NO. 2, and the sequence of the downstream primer is shown as SEQ ID NO. 3.

By adopting the primer group, the miRNA related to the follicular development of the laying duck can be detected by a stem-loop method.

Further, in some embodiments of the invention, the kit contains a reagent for reverse transcription, a reverse transcription primer, a specific primer, an internal reference primer, and a fluorescent quantitative PCR reaction reagent.

The internal reference primers comprise an upstream primer and a downstream primer of a U6 internal reference gene, wherein the sequence of the upstream primer is shown as SEQ ID NO. 6, and the sequence of the downstream primer is shown as SEQ ID NO. 7.

As a preferred embodiment of the kit, the kit also comprises an RNA extraction reagent, a reverse transcription reaction system and a fluorescent quantitative PCR reaction system, wherein the fluorescent quantitative PCR reaction system comprises THUNDERBIRD SYBR qPCR Mix, ddH₂O, cDNA obtained by reverse transcription of the reverse transcription reaction system and the detection primer pair.

As the specific embodiment, the fluorescent quantitative PCR reaction system comprises 10 mu L of THUNDERBIRD SYBR qPCR Mix, 0.4 mu L of 10uM egg-laying duck apla-mir-25-42 upstream primer, 0.4 mu L of 10uM egg-laying duck apla-mir-25-42 downstream primer, 2.0 mu L of cDNA template to be detected and ddH₂O7.2. mu.L, total volume 20. mu.L.

In a seventh aspect of the invention, there is provided a method of using the kit, comprising the steps of:

step 1, extracting RNA in a sample to be detected;

step 2, reverse transcription is carried out on apla-mir-25-42 under the action of reverse transcriptase by utilizing a reverse transcription primer to generate cDNA;

step 3, performing fluorescent quantitative PCR amplification on the cDNA of the sample to be detected by using the primers (such as the upstream primer shown in SEQ ID NO:2, the downstream primer shown in SEQ ID NO:3 and the LOOP primer shown in SEQ ID NO: 5), and adopting 2^-ΔΔCTCalculating and obtaining the relative expression quantity of the apla-mir-25-42 in the follicle samples from different sources by the method;

and 4, judging whether the egg-laying duck apla-mir-25-42 and the expression condition of the egg-laying duck apla-mir-25-42 exist in the sample to be detected according to the PCR amplification product and the relative gene expression quantity.

As the above embodiment, the conditions of the fluorescent quantitative PCR reaction in the step 3 are 98 ℃ and 10 s; 94 ℃, 15s, 54 ℃, 15s, 60 ℃, 15s, 40 cycles; 95 ℃ for 10 s; 65 ℃, 60s, 97 ℃, 1s, 1 cycle.

In the eighth aspect of the invention, a mimic (namely mimic) of miRNA apla-mir-25-42 is provided, and the sequence of the mimic is shown as SEQ ID NO. 1 and SEQ ID NO. 8.

The ninth aspect of the invention provides the application of the apla-mir-25-42 simulant in detecting the proliferation and differentiation of the follicular granular cells of the laying ducks.

Through research, the inventor finds that when the mimic transfection of the miRNA apla-mir-25-42 of the laying duck enters the follicular granular cells of the laying duck, compared with an NC group, the expression level of a cell proliferation marker gene CyclinB2 in the mimic group is remarkably increased (P <0.01), and the expression level of an apoptosis marker gene BCL2 is remarkably reduced (P <0.01), so that the miRNA can be used as a molecular marker for detecting the proliferation and differentiation conditions of the cells in the follicular granular cells of the laying duck.

In the tenth aspect of the invention, the invention provides a method for regulating the follicular development of laying ducks, which comprises the step of applying an agent containing RNA as shown in SEQ ID NO. 1 and SEQ ID NO. 8 as an active ingredient to the follicular granulosa cells of the target laying ducks.

Preferably, the control of the follicular development of the laying duck is to promote the proliferation of the follicular granular cells of the laying duck or inhibit the apoptosis of the follicular granular cells of the laying duck.

The miRNA mimic shown in SEQ ID NO. 1 and SEQ ID NO. 8 is used for transfecting the laying duck follicular granular cells, and surprisingly found that the expression level of a cell proliferation marker gene CyclinB2 is increased, and the expression level of an apoptosis marker gene BCL2 is reduced, and the result shows that the miRNA shown in SEQ ID NO. 1 can be used for regulating and controlling the development of the laying duck follicular.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the relative quantitative expression result of the laying duck apla-mir-25-42 provided by the invention in white follicles and yellow follicles of the laying duck;

FIG. 2 shows the change of the expression of the apla-mir-25-42 in the group overexpressing the apla-mir-25-42 and the control group after the apla-mir-25-42 of the laying ducks in example 2 is overexpressed;

FIG. 3 shows the change of expression of key genes for cell proliferation and apoptosis in the group overexpressing apla-mir-25-42 and the control group after overexpressing the apla-mir-25-42 in example 2.

FIG. 4 is a schematic diagram of the structure of a pmirGLO vector.

FIG. 5 is a dual luciferase assay demonstrating the binding of apla-mir-25-42 to the 3' -UTR of TGFB 2.

Detailed Description

Example 1 obtaining PCR of miRNA apla-mir-25-42 from laying duck and fluorescent quantitative detection kit and method thereof

One, egg duck apla-mir-25-42

The corresponding nucleotide sequence of the laying duck apla-mir-25-42 is shown as SEQ ID NO:1, and the laying duck apla-mir-25-42 is a new miRNA molecule obtained by carrying out a large amount of bioinformatics analysis and screening on the sequencing results of two groups of follicle tissue samples at different development periods based on the sequencing result of the earlier-completed laying duck follicle tissue complete transcriptome. The applicant names the duck apla-mir-25-42.

Fluorescent quantitative PCR detection primer pair of egg-laying duck miRNA apla-mir-25-42, detection kit and method thereof

In the embodiment, a fluorescent quantitative PCR detection primer pair is designed, and the objective existence of the laying duck apla-mir-25-42 in the follicular granular cells of the laying duck is identified and identified. The method for identifying and authenticating the mobile terminal comprises the following steps:

1. sample collection

Collecting 3 parts of each of white follicle and yellow follicle samples of laying ducks in the egg production peak period, cleaning the white follicle and the yellow follicle samples with PBS, puncturing the follicles with a needle to extrude follicular fluid, putting the follicular fluid into an EP (European patent) tube with 1.5mL without enzyme, marking, and then putting the follicular fluid into a liquid nitrogen or refrigerator at minus 80 ℃ for storage.

2. Sample total RNA extraction

Extracting total RNA of follicular tissue by using a traditional TRIzol and chloroform method, which comprises the following steps:

1) grinding by using liquid nitrogen, namely putting 50-100mg of tissue sample into a mortar, pouring a little liquid nitrogen, quickly grinding, adding a little liquid nitrogen when the liquid nitrogen is completely volatilized until the tissue sample is ground into powder, transferring the tissue sample into a 1.5mL centrifuge tube, adding 1mL Trizol, testing the temperature, and standing for 5min to fully crack the tissue sample;

2) centrifuging at 12000g at 4 deg.C for 10min, and transferring the supernatant into a clean 1.5mL centrifuge tube without RNAase;

3) adding 0.2ml chloroform into 1ml homogenate for 15s, and standing at room temperature for 15 min;

4) centrifuging at 12000g at 4 deg.C for 10 min;

5) carefully sucking the upper layer colorless solution out, adding 0.5 times of isopropanol into another 1.5ml centrifuge tube without RNAase, mixing uniformly, testing the temperature and standing for 5-10 min;

6) centrifuging at 12000g at 4 deg.C for 10min, removing supernatant, and precipitating RNA at the bottom of the tube;

7) adding 1mL of 75% ethanol, gently oscillating the centrifugal tube, and suspending and precipitating;

8) centrifuging at 8000g at 4 deg.C for 5min, and removing supernatant;

9) adding 1mL of absolute ethyl alcohol, gently oscillating the centrifugal tube, and suspending and precipitating;

10) centrifuging at 4 deg.C 8000g for 5min, discarding supernatant, and air drying at room temperature;

11) RNA was dissolved in 30-50. mu.L of RNAase-free deionized water and stored at-80 ℃ for further use.

3. RNA sample quality detection assay

RNA sample concentration and OD value were measured using a NanoDrop 1000 microspectrophotometer, and RNA integrity was measured using 1% agarose gel electrophoresis

4. Reverse transcription

Reverse transcription kit using Takara

The RT reagent Kit was reverse transcribed with the instructions of gDNA Eraser to obtain a cDNA template, wherein the reverse transcription primer sequence was apla-mir-25-42-LOOP (shown in SEQ ID NO: 5).

1) Reaction for removing genomic DNA

TABLE 1

The reaction conditions were as follows:

2min at 42 ℃; storing at 4 ℃.

2) Reverse transcription reaction

The reaction solution preparation process was performed on ice.

TABLE 2

The reaction conditions were as follows:

37 ℃ for 15 min; 85 ℃ for 5 sec; storing at 4 ℃.

5. Fluorescent quantitative PCR reaction

The nucleotide sequences of the primer for fluorescent quantitative detection of the apla-mir-25-42 are shown as SEQ ID NO. 2 and SEQ ID NO. 3, and the nucleotide sequences of the primer for fluorescent quantitative detection of the internal reference gene are shown as SEQ ID NO. 6 and SEQ ID NO. 7.

Fluorescent quantitative PCR was performed using the procedure described in the Takara Real time kit THUNDERBIRD SYBR qPCR Mix, and the reaction system was as follows (20. mu.L):

TABLE 3

Using Roche

Carrying out fluorescent quantitative PCR by a 96SW 1.1 three-step method under the amplification conditions of 98 ℃ and 10 s; 94 ℃, 15s, 54 ℃, 15s, 60 ℃, 15s, 40 cycles; 95 ℃ 10s, 65 ℃ 60s, 97 ℃ 1s, 1 cycle.

Data were derived after the reaction was complete and results were used 2^-ΔΔCTThe method is used for analysis. Results of the analysis were plotted using Graphad Prism software.

The results of the expression of the apla-mir-25-42 in the tissues of the white follicles and the yellow follicles are shown in the figure 1, and the results show that, firstly, the expression of the apla-mir-25-42 exists in the white follicles and the yellow follicles, and the laying duck apla-mir-25-42 objectively exists in the granulosa cells of the laying duck follicles. Second, the expression level of apla-mir-25-42 in white follicles was significantly higher than in yellow follicles (P < 0.01). Thus, it can be seen that apla-mir-25-42 has an important correlation with the development of follicles.

Example 2 application of apla-mir-25-42 as molecular marker in detecting proliferation and differentiation of follicular granulosa cells of laying ducks

First, the separation of follicular granular cells of laying duck

The method for separating the follicular granular cells of the laying duck comprises the following steps:

1. selecting laying ducks in the egg laying peak period, and killing the ducks by jugular vein bloodletting; taking out the whole ovarian tissue, and placing the whole ovarian tissue in a sterile culture dish filled with precooled PBS;

2. washing blood stain with PBS buffer solution containing double antibody, and rinsing for 3 times;

3. transferring the rinsed follicles into a plate filled with precooled PBS buffer solution, and stripping the outer membranes, connective tissues and vascular networks of the follicles;

4. cutting a 1-2cm incision on the surface of the follicle (action is quick) with a scalpel, releasing yolk, and rinsing the residual yolk solution with PBS buffer solution to obtain the residual follicle membrane, i.e. basement membrane and follicle granule cell layer;

5. shearing the rinsed follicular membrane as much as possible, placing in a 15mL centrifuge tube, adding 4mL culture medium, repeatedly blowing with 1mL pipette for 1min, centrifuging at 4 deg.C and 1000rpm, and removing supernatant;

6. adding 4mL of 0.2% collagenase II into the precipitate, resuspending the precipitate, and digesting for 30min by placing the precipitate in a constant temperature shaker at 37 ℃ and 80 rpm;

7. at the end of digestion, 4mL of M199 complete medium (containing 10% serum) was added to stop digestion; filtering with 200 mesh stainless steel sieve, washing the mesh with 2mL M199 complete culture medium, collecting filtrate, and centrifuging at 4 deg.C 1000rpm for 10 min;

8. discarding the supernatant, adding 10mL of M199 complete medium, and centrifuging at 4 ℃ and 1000rpm for 10 min;

9. the supernatant was discarded, resuspended in M199 complete medium (containing 10% FBS and 1% double antibody), and the cell density was determined by adjusting the cell density of the suspension to 1X 10⁶one/mL of the cells were inoculated in 6-well plates and incubated at 37 ℃ with 5% CO₂Carrying out static culture in an incubator;

10. after the granulosa cells are cultured for 24 hours, the adherent cells can be used for further research by replacing fresh M199 culture medium containing fetal bovine serum.

Second, cell transfection

1. 125 mu.L of Opti-MEM culture medium is respectively taken to dilute the synthesized apla-mir-25-42mim (namely SEQ ID NO:1 and SEQ ID NO:8) and NC;

2. diluting 250 μ L of Opti-MEM medium

3000 reagent, fully and uniformly mixing;

3. adding 125 μ L of the mixture in (2) to the two tubes of the mixture in (1), and incubating at room temperature for 5 min;

4. 250 μ L of the mixture was transferred to the cells.

Each experimental group was set with 3 replicate wells, and transfected cells were grouped (i) transfection NC, (ii) transfection apla-mir-25-42. After each transfection group transfects cells for 24h, the cells are digested by Trizol, and total RNA of the cells is extracted for quantitative analysis of fluorescence after overexpression.

Third, fluorescent quantitative analysis

1. Extraction of Total RNA from cells

Total RNA was extracted from the cells according to the method for extracting total RNA described in example 1.

2. Reverse transcription

The reverse transcription was performed as in example 1.

3. Fluorescent quantitative PCR reaction

(1) The fluorescent quantitative PCR reaction conditions and method are the same as example 1, wherein the nucleotide sequences of the apla-mir-25-42 fluorescent quantitative primer are shown as SEQ ID NO. 2 and SEQ ID NO. 3, and the amplification conditions are 98 ℃ and 10 s; 94 ℃, 15s, 54 ℃, 15s, 60 ℃, 15s, 40 cycles; 1 cycle of 95 ℃ for 10s, 65 ℃ for 60s, 97 ℃ for 1 s;

(2) the nucleotide sequence of the fluorescent quantitative primer of the cell proliferation marker gene CyclinB2 is shown as SEQ ID NO. 9 and SEQ ID NO. 10, and the amplification conditions are 98 ℃ and 10 s; 94 ℃, 15s, 56 ℃, 15s, 60 ℃, 15s, 40 cycles; 1 cycle of 95 ℃ for 10s, 65 ℃ for 60s, 97 ℃ for 1 s;

(3) the apoptosis marker gene BCL2 fluorescent quantitative primer has nucleotide sequences shown in SEQ ID NO. 11 and SEQ ID NO. 12, and the amplification conditions are 98 deg.C and 10 s; 94 ℃, 15s, 54 ℃, 15s, 60 ℃, 15s, 40 cycles; 1 cycle of 95 ℃ for 10s, 65 ℃ for 60s, 97 ℃ for 1 s;

(4) the nucleotide sequences of the internal reference gene fluorescent quantitative primer are shown as SEQ ID NO. 6 and SEQ ID NO. 7, and the amplification conditions are 98 ℃ and 10 s; 94 ℃, 15s, 54 ℃, 15s, 60 ℃, 15s, 40 cycles; 95 ℃ 10s, 65 ℃ 60s, 97 ℃ 1s, 1 cycle.

4. Analysis of results

Data were derived after the reaction was complete and results were used 2^-ΔΔCTThe method is used for analysis. Results of the analysis were plotted using Graphad Prism software. The analysis results of the expression quantity of the apla-mir-25-42 and the expression quantity of the cell proliferation and apoptosis marker genes after the egg-laying duck apla-mir-25-42 is transfected are shown in figures 2-3.

As can be seen from FIG. 2, after the apla-mir-25-42 is over-expressed, compared with the NC group, the expression level of the apla-mir-25-42 after the egg-laying duck apla-mir-25-42 is over-expressed is remarkably higher than that of the NC group (P < 0.01).

As can be seen from FIG. 3, after the laying duck apla-mir-25-42 is over-expressed, compared with the NC group, after the laying duck apla-mir-25-42 is over-expressed, the expression level of the cell proliferation marker gene CyclinB2 is remarkably increased (P <0.01), and the expression level of the apoptosis marker gene BCL2 is remarkably reduced (P <0.01), which indicates that the miRNA promotes the proliferation of cells. Therefore, the miRNA can be used as a molecular marker for detecting the cell proliferation and differentiation condition in the follicular granular cells of the laying ducks.

In conclusion, the synthesized laying duck apla-mir-25-42mim can stably express the laying duck apla-mir-25-42, can be used as a detection index to reflect the expression level of the laying duck apla-mir-25-42, and can be applied to detection of cell proliferation and differentiation conditions in the laying duck follicular granular cells.

Example 3 design and construction of duckling apla-mir-25-42 target Gene TGFB2 Dual-luciferase reporter Gene detection System

Firstly, performing correlation analysis on a whole transcriptome sequencing result aiming at a selected miRNA apla-mir-25-42, screening a target gene TGFB2 interacting with the apla-mir-25-42 from the mRNA sequencing result, and obtaining a binding site between the apla-mir-25-42 and TGFB2 as ATCCCA by utilizing miRBase analysis.

Subsequently, the 3' -UTR sequence of TGFB2 (i.e., the inhibitor of apla-mir-25-42) was obtained from NCBI, ATCCCA in the inhibitor of apla-mir-25-42 was mutated to CAGGCGC by mutation, and the above two gene fragments were cloned into pmirGLO vector to obtain the corresponding vector containing the inhibitor sequence of apla-mir-25-42 (pmirGLO-TGFB2-wt) and the vector containing the inhibitor mutation sequence of apla-mir-25-42 (pmirGLO-TGFB2-mut), respectively. The vector construction specifically comprises the following steps:

(1) double digestion treatment of pmirGLO vector

The schematic structure of the pmirGLO vector is shown in FIG. 4, and the vector contains restriction sites for restriction enzymes PmeI and XhoI. The vector is subjected to double enzyme digestion by two enzymes PmeI and XhoI, the enzyme digestion reaction system is as follows, and the total volume of the system is 50 mu L:

TABLE 4

The reaction conditions are that the enzyme digestion is carried out for 6h at 37 ℃, and then the enzyme digestion product is purified and recovered.

(2) Obtaining and verifying of TGFB2 gene 3' UTR segment before and after mutation of laying duck

The primer is designed to amplify the TGFB2 gene 3 'UTR fragments before and after mutation aiming at the sequences before and after mutation of the binding site of the apla-mir-25-42 in the egg-laying duck TGFB2 gene 3' UTR, the nucleotide sequences of the adopted amplification primers before and after mutation are respectively shown as SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16, and the reaction system is as follows (20 mu L).

TABLE 5

The amplification conditions are 95 ℃ and 300 s; 94 ℃, 30s, 56 ℃, 30s, 72 ℃, 30s, 30 cycles; 72 ℃ and 600 s. And detecting the PCR product obtained by amplification by using 1% agarose gel electrophoresis and carrying out sequencing verification.

(3) Double enzyme digestion of TGFB2 gene 3' UTR fragment before and after mutation

Purifying the PCR product which is verified to be correct, and carrying out double enzyme digestion on the recovered PCR product by using two enzymes PmeI and XhoI, wherein the enzyme digestion reaction system is as follows, and the total volume of the system is 50 mu L:

TABLE 6

(3) Construction of pmirGLO-TGFB2-wt and pmirGLO-TGFB2-mut vectors

The TGFB2 gene 3' UTR fragment amplified by PCR and subjected to double enzyme digestion is connected with a vector subjected to double enzyme digestion, and a connection reaction system is as follows (the total volume is 10 mu L):

TABLE 7

The reaction conditions were 16 ℃ overnight for ligation.

Transforming the ligation product into competent cells, and performing colony PCR identification and sequencing verification on the positive clone, wherein the sequencing results are shown as SEQ ID NO.4 and SEQ ID NO. 17; the successfully constructed recombinant vectors were named pmirGLO-TGFB2-wt and pmirGLO-TGFB2-mut, respectively.

Example 4 mechanism of Apla-mir-25-42 for promoting the proliferation of follicular granulosa cells of laying ducks

Separating follicular granulosa cells of egg-laying duck at 5 × 10⁴Perwell were inoculated in 24-well plates and co-transfected with pmarGLO, pmirGLO-TGFB2-wt, pmirGLO-TGFB2-mut, respectively, with apla-mir-25-42 mimic. Cells were harvested 24h after co-transfection and used

The Reporter Assay System detects the relative fluorescence activity of the fluorescent carrier in the microplate reader, and the detection result is shown in FIG. 5. The result shows that after the wild-type vectors of the apla-mir-25-42 and the TGFB2 are co-transfected, the dual luciferase activity is obviously reduced (P is less than 0.05) compared with that of the mutant and empty vector co-transfer group, which indicates that the mimic of the apla-mir-25-42 can be combined with the 3 'UTR of the TGFB2 gene, and the result indicates that the apla-mir-25-42 can inhibit the expression of the TGFB2 through targeting the 3' UTR combined with the TGFB2 gene, thereby promoting the proliferation of the follicular granular cells of the laying ducks.

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

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<213> Artificial sequence

<400> 4

gtgctttgta tattgttcat cattatgaca taagctacct gactccattt ggtttttgtt 60

ttataaaagg atggattaaa gcgctctttc ctcccctcct tctctccctc ctcgcccatc 120

cccattttta ttttttcttt tggcgttaga cattcaaaca gatgggcagg gagcgaggca 180

ttgcaagaag agacgtccca gcctggctga ggcaaggcga agccttgcgg agggtgacag 240

<210> 5

<211> 44

<212> DNA

<213> Artificial sequence

<400> 5

ctcaactggt gtcgtggagt cggcaattca gttgagaagt aatg 44

<210> 6

<211> 17

<212> DNA

<213> Artificial sequence

<400> 6

ctcgcttcgg cagcaca 17

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<400> 7

aacgcttcac gaatttgcgt 20

<210> 8

<211> 22

<212> RNA

<213> Artificial sequence

<400> 8

aaguaaugca acaaaucccc ac 22

<210> 9

<211> 18

<212> DNA

<213> Artificial sequence

<400> 9

ttacaacaag aggaacgc 18

<210> 10

<211> 18

<212> DNA

<213> Artificial sequence

<400> 10

tccataggga caggagac 18

<210> 11

<211> 18

<212> DNA

<213> Artificial sequence

<400> 11

acggctctcg ctcctgct 18

<210> 12

<211> 18

<212> DNA

<213> Artificial sequence

<400> 12

cggttgacgc tctccacg 18

<210> 13

<211> 54

<212> DNA

<213> Artificial sequence

<400> 13

atcgccgtgt aattctagtt gtttaaacgt gctttgtata ttgttcatca ttat 54

<210> 14

<211> 46

<212> DNA

<213> Artificial sequence

<400> 14

gcaggtcgac tctagactcg aggctagcct gtcaccctcc gcaagg 46

<210> 15

<211> 54

<212> DNA

<213> Artificial sequence

<400> 15

gtttaaacgct ctttcctccc ctccttctct ttattttttc ttttggcgtt agac 54

<210> 16

<211> 30

<212> DNA

<213> Artificial sequence

<400> 16

cctcgaggga gaaggagggg aggaaagagc 30

<210> 17

<211> 240

<212> DNA

<213> Artificial sequence

<400> 17

gtgctttgta tattgttcat cattatgaca taagctacct gactccattt ggtttttgtt 60

ttataaaagg atggattaaa gcgctctttc ctcccctcct tctctccctc ctcgccccag 120

gcgcttttta ttttttcttt tggcgttaga cattcaaaca gatgggcagg gagcgaggca 180

ttgcaagaag agacgtccca gcctggctga ggcaaggcga agccttgcgg agggtgacag 240

Claims

1. An miRNA related to the follicular development of laying ducks is characterized in that the miRNA is apla-mir-25-42, and the nucleotide sequence of the miRNA is shown as SEQ ID NO. 1.

2. The application of miRNA related to the follicular development of laying duck as claimed in claim 1 as a molecular marker in detecting the proliferation and differentiation status of follicular granulosa cells of laying duck.

3. The primer for detecting the miRNA marker related to the follicular development of the laying duck as claimed in claim 1.

4. The primer according to claim 3, wherein the primer comprises an upstream primer shown in SEQ ID NO. 2, a downstream primer shown in SEQ ID NO. 3, and a LOOP primer shown in SEQ ID NO. 5.

5. An inhibitor for inhibiting the miRNA related to the follicular development of the laying duck in claim 1 from binding to a target gene, wherein the target gene is TGFB2 gene, and the binding site of the inhibitor to the miRNA is ATCCCA;

the inhibitor is selected from a segment of about 240bp in length in the 3' UTR of the TGFB2 gene;

the nucleotide sequence of the inhibitor is shown as SEQ ID NO. 4.

6. The inhibitor according to claim 5, wherein a restriction enzyme site sequence is added to each of the 5 'end and the 3' end of the inhibitor.

7. The inhibitor of claim 6, wherein the restriction enzyme cleavage site sequence produces a sticky end upon cleavage.

8. The inhibitor according to claim 7 wherein the restriction site sequence is a nucleotide sequence corresponding to the PmeI restriction site and the XhoI restriction site, respectively.

9. The inhibitor of claim 8, wherein the PmeI cleavage site is located at the 5 'end and the XhoI cleavage site is located at the 3' end.

10. A recombinant expression vector comprising the inhibitor of any one of claims 5-9.

11. The recombinant expression vector of claim 10, wherein the marker gene in the recombinant expression vector is a dual luciferase reporter gene.

12. The recombinant expression vector according to claim 11, wherein the recombinant expression vector is obtained by ligating the inhibitor into a reporter vector pmirGLO.

13. Use of the miRNA of claim 1 or the inhibitor of any one of claims 5-9 for proliferation and differentiation of follicular granulosa cells of laying ducks.

14. A method for regulating the follicular development of laying ducks comprises administering an agent containing RNA as shown in SEQ ID NO 1 and SEQ ID NO 8 as an active ingredient to follicular granulosa cells of a target laying duck;

the control of the follicular development of the laying duck is to promote the proliferation of the follicular granular cells of the laying duck or inhibit the apoptosis of the follicular granular cells of the laying duck.