CN110295170B - Long-chain RNA Lnc-13814 for regulating follicular development of laying duck and application thereof - Google Patents
Long-chain RNA Lnc-13814 for regulating follicular development of laying duck and application thereof Download PDFInfo
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Abstract
The invention provides a long-chain RNA Lnc-13814 for regulating and controlling follicular development of laying ducks and application thereof. The cDNA sequence corresponding to the long-chain RNA Lnc-13814 related to the follicular development of the laying duck is shown as SEQ ID NO: 1 is shown in the specification; the invention also provides a fluorescent quantitative PCR detection primer pair and a detection kit of the long-chain RNA Lnc-13814, wherein the detection primer pair is shown as SEQ ID NO: 2 and SEQ ID NO: 3; the invention also provides the long-chain RNA Lnc-13814 overexpression vector and a construction method thereof. The invention discovers that the long-chain RNA Lnc-13814 has the function of promoting the proliferation of follicular granular cells of the laying ducks for the first time. Has important significance for researching the genetic essence of the breeding character of the laying duck and improving the reproductive capacity of the laying duck.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a long-chain RNA Lnc-13814 for regulating and controlling follicular development of laying ducks and application thereof.
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. 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.
Long non-coding RNAs (LncRNAs) are RNAs molecules with lengths of more than 200nt, lack of specific complete open reading frames and have no or few protein coding functions. Research has shown that LncRNAs play an important role in physiological and pathological processes such as cell proliferation, cell cycle, apoptosis, invasion and migration, and participate in regulation and control of important life activities such as organism growth, development, aging and death by widely regulating gene expression at epigenetic, transcriptional and post-transcriptional levels. Research in recent years finds that some LncRNA molecules have important significance in human tumor diagnosis and treatment; research on chickens finds that LncRNA has important regulation and control effects on intramuscular adipocyte differentiation, meat quality, lipid metabolism, carcass traits, reproductive performance and the like.
However, no report is found on the research on the tissue-specific LncRNA of the laying duck, particularly the research on the LncRNA of the follicle of the laying duck and the regulation mechanism thereof. Therefore, the research level and the integrity of the laying duck follicle LncRNA are urgently needed to be improved, so that a scientific theoretical basis is provided for the research of the laying duck genome and the non-coding RNA thereof, the control mechanism of the laying duck follicle development is further understood, and a theoretical support is provided for improving the reproductive performance of the laying duck.
Disclosure of Invention
The invention aims to overcome the blank of research in the prior art and provides a long-chain RNA Lnc-13814 for regulating and controlling the follicular development of laying ducks and application thereof.
In order to achieve the purpose, the invention is realized by the following scheme:
in the first aspect of the invention, the invention provides a long-chain RNA Lnc-13814 for regulating follicular development of an laying duck, wherein a cDNA sequence corresponding to LncRNA13814 of the laying duck is shown as SEQ ID NO: 1 is shown.
The long-chain RNA Lnc-13814 for regulating the development of the follicles of the laying ducks is a new long-chain RNA molecule obtained by analyzing and screening a large amount of bioinformatics on the sequencing results of two groups of follicle tissue samples at different development periods on the basis of the sequencing results of the full transcriptome of the follicular tissue of the laying ducks completed by the applicant at the previous stage. The applicant names the cDNA sequence of the long-chain RNA Lnc-13814 of the laying duck, and the length of the cDNA sequence corresponding to the long-chain RNA Lnc-13814 of the laying duck is 974 bp.
In the second aspect of the invention, the application of the long-chain RNA Lnc-13814 for regulating the follicular development of the laying duck as a molecular marker in identifying the follicular development condition of the laying duck is provided.
Through research, the applicant finds that the expression level of the long-chain RNA Lnc-13814 for regulating the development of the follicles of the laying ducks in white follicles of the laying ducks is obviously higher than that of the white follicles of the laying ducks, so that the long-chain RNA Lnc-13814 of the laying ducks can be used as a molecular marker for identifying the development conditions of the follicles of the laying ducks.
In a third aspect of the invention, the application of the long-chain RNA Lnc-13814 for regulating the follicular development of the laying duck as a molecular marker in detecting the proliferation and differentiation conditions of cells in follicular granulosa cells of the laying duck is provided.
Through research, the applicant finds that an overexpression vector consisting of long-chain RNA Lnc-13814 molecules for regulating the development of follicles of laying ducks is transfected into the follicular granular cells of the laying ducks, compared with an empty vector group, after the long-chain RNA Lnc-13814 genes of the laying ducks are overexpressed, the expression level of a marker gene BCL2 for inhibiting apoptosis is greatly reduced (P <0.01), the expression level of a marker gene FAS for promoting apoptosis is greatly increased (P <0.01), and therefore the LncRNA can be used as a molecular marker for detecting the apoptosis condition in the follicular granular cells of the laying ducks; after the long-chain RNA Lnc-13814 is over-expressed, the apoptosis number is increased, which shows that the long-chain RNA Lnc-13814 has promotion effect on the apoptosis of the follicular granular cells of the laying duck, thereby further proving that the LncRNA can be used as a molecular marker for detecting the apoptosis condition of the follicular granular cells of the laying duck.
In the fourth aspect of the invention, a fluorescent quantitative PCR detection primer pair of long-chain RNA Lnc-13814 gene for regulating and controlling the follicular development of laying ducks is provided, which comprises:
the upstream primer long-chain RNA Lnc-13814-F is shown as SEQ ID NO: 2;
the long-chain RNA Lnc-13814-R of the downstream primer is shown as SEQ ID NO: 3.
In the fifth aspect of the invention, the fluorescent quantitative PCR detection kit for the long-chain RNA Lnc-13814 gene for regulating and controlling the follicular development of the laying duck is provided, and comprises the fluorescent quantitative PCR detection primer pair.
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 above embodiment, the fluorescent quantitative PCR reaction system is as follows: 10 mu L of THUNDERBIRD SYBR qPCR Mix, 0.4 mu L of each of 10uM egg duck long-chain RNA Lnc-13814 upstream primer and downstream primer, 2.0 mu L of cDNA template to be detected, and ddH2O7.2. mu.L, total volume 20. mu.L.
In a sixth aspect of the invention, there is provided a method of using the kit, comprising the steps of:
and 3, judging whether the egg-laying duck long-chain RNA Lnc-13814 gene and the expression condition of the egg-laying duck long-chain RNA Lnc-13814 gene 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 step 2 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 seventh aspect of the invention, a long-chain RNA Lnc-13814 gene overexpression vector for regulating and controlling the follicular development of the laying duck is provided, and the vector comprises a cDNA sequence corresponding to the long-chain RNA Lnc-13814 of the laying duck.
The invention has the beneficial effects that:
1. the invention provides a fluorescent quantitative PCR detection primer pair and a detection kit of long-chain RNA Lnc-13814 for regulating follicular development of laying ducks, wherein a specific primer is designed according to a gene sequence of the long-chain RNA Lnc-13814 of the laying ducks, the expression of LncRNA is detected by using fluorescent quantitative PCR, and then whether the long-chain RNA Lnc-13814 gene of the laying ducks exists in a sample to be detected and the expression condition of the long-chain RNA Lnc-13814 gene of the laying ducks are judged according to PCR amplification products and relative gene expression quantity.
2. The invention provides a long-chain RNA Lnc-13814 for regulating and controlling follicular development of laying ducks, (1) application of the LncRNA as a molecular marker in identifying follicular development of the laying ducks: the invention utilizes the fluorescent quantitative PCR detection primer pair and the detection kit, can accurately detect different expression conditions of the long-chain RNA Lnc-13814 in follicular tissues at different development stages, thereby analyzing and finding that the long-chain RNA Lnc-13814 gene of the laying duck is related to the follicular development of the laying duck for the first time, making up the existing vacancy in the field, providing theoretical basis and scientific basis for analyzing the genetic mechanism of the follicular development of the laying duck, and having important significance for researching the genetic essence of the reproductive traits of the laying duck and improving the reproductive capacity of the laying duck. (2) The LncRNA is used as a molecular marker for detecting the apoptosis condition in the follicular granular cells of the laying duck: the invention uses the egg duck long-chain RNA Lnc-13814 gene overexpression vector to stably express the egg duck long-chain RNA Lnc-13814 gene, the detection index reflects the expression quantity of the egg duck long-chain RNA Lnc-13814 gene, compared with an empty vector group, after the egg duck long-chain RNA Lnc-13814 gene is overexpressed, the expression quantity of the apoptosis-inhibiting marker gene BCL2 is greatly reduced (P <0.01), the expression quantity of the apoptosis-promoting marker gene FAS is greatly increased (P <0.01), thereby obtaining that the LncRNA can be used as a molecular marker to be applied to detecting the apoptosis condition in the egg duck follicular granular cells; after the long-chain RNA Lnc-13814 is over-expressed, the apoptosis number is increased, which shows that the long-chain RNA Lnc-13814 has promotion effect on the apoptosis of the follicular granular cells of the laying duck, thereby further proving that the LncRNA can be used as a molecular marker for detecting the apoptosis condition of the follicular granular cells of the laying duck.
3. The egg-laying duck long-chain RNA Lnc-13814 gene overexpression vector provided by the invention contains a full-length sequence fragment of a long-chain RNA Lnc-13814 gene, can stably express the long-chain RNA Lnc-13814 of an egg-laying duck, can reflect the expression quantity of the egg-laying duck long-chain RNA Lnc-13814 gene through fluorescent quantitative detection, and can be applied to the related technical field; the construction method of the egg-laying duck long-chain RNA Lnc-13814 gene overexpression vector provided by the invention is simple to operate, is quick and easy to obtain, can accurately obtain the recombinant vector with the target gene through screening, and the prepared recombinant vector can stably express the target gene.
Drawings
FIG. 1 is a schematic structural diagram of pcDNA3.1 vector in an over-expression vector of long-chain RNA Lnc-13814 gene of laying duck provided in example 2 of the present invention;
FIG. 2 is a diagram showing the result of colony PCR electrophoresis of the long-chain RNA Lnc-13814 gene overexpression vector of the laying duck provided in example 2 of the present invention; the reference marks indicate that a lane M in the figure is DL2000DNA marker, and lanes 1 and 2 are long-chain RNA Lnc-13814 overexpression vector bacterial liquid amplification fragments;
FIG. 3 is the fluorescent quantitative PCR detection primer pair of the long-chain RNA Lnc-13814 gene of the laying duck and the relative quantitative expression result of the long-chain RNA Lnc-13814 gene of the laying duck in the detection kit thereof, which are provided by the embodiment 2 of the invention;
FIG. 4 shows the expression changes of the long-chain RNA Lnc-13814 gene in the over-expression vector group and the empty vector group after the long-chain RNA Lnc-13814 gene of the laying duck is over-expressed in example 3;
FIG. 5 shows the change of expression of key genes for cell proliferation and apoptosis in an over-expression vector group and an empty vector group after the long-chain RNA Lnc-13814 of the laying duck is over-expressed in example 3;
FIG. 6 shows the apoptosis changes in the over-expression vector group and the empty vector group after the long-chain RNA Lnc-13814 is over-expressed in example 3.
Detailed Description
Example 1
Egg duck long-chain RNA Lnc-13814 gene
The cDNA sequence corresponding to the long-chain RNA Lnc-13814 of the laying duck is shown as SEQ ID NO: 1, the egg-laying duck long-chain RNA Lnc-13814 is a novel LncRNA molecule obtained by carrying out a large amount of bioinformatics analysis and screening on the sequencing results of two groups of follicle tissue samples in different developmental stages based on the sequencing results of the earlier-completed egg-laying duck follicle tissue complete transcriptome. The applicant names the cDNA sequence of the long-chain RNA Lnc-13814 of the laying duck, and the length of the cDNA sequence corresponding to the long-chain RNA Lnc-13814 of the laying duck is 974 bp.
Preparation of long-chain RNA Lnc-13814 gene full-length sequence fragment of egg-laying duck
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 values were determined using a NanoDrop 1000 microspectrophotometer, and RNA integrity was checked using 1% agarose gel electrophoresis.
4. Reverse transcription
Reverse transcription kit using Takara
The RT reagent Kit with the instructions of gDNA Eraser reverse transcribes to obtain cDNA template.
(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: 15min at 37 ℃; 85 ℃ for 5 sec; storing at 4 ℃.
5. Full-length fragment for amplifying long-chain RNA Lnc-13814 gene sequence of laying duck by using cDNA obtained by reverse transcription as template
(1) Primer design and Synthesis
According to the full-length segment of the long-chain RNA Lnc-13814 gene sequence of the laying duck in the sequencing result of the complete transcriptome, a primer is designed by adopting Premier Premier 5.0 software, and the amplification primer sequence of the long-chain RNA Lnc-13814 gene of the laying duck is shown as SEQ ID NO: 4 and SEQ ID NO: 5 is shown in the specification; the above primers were synthesized by Beijing Olympic Biotechnology Ltd.
(2) The obtained target fragment egg duck long-chain RNA Lnc-13814 gene PCR product is directly sequenced, and the sequencing result is completely consistent with the expected result.
Example 2 fluorescent quantitative detection kit and method for long-chain RNA Lnc-13814 gene of laying duck
In the embodiment, a fluorescent quantitative PCR detection primer pair is designed, and the objective existence of the long-chain RNA Lnc-13814 of the laying duck described in the embodiment 1 in the follicular granular cells of the laying duck is 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
The specific steps of extracting total RNA of follicular tissue by using traditional TRIzol and chloroform method are the same as example 1.
3. RNA sample quality detection assay
RNA sample concentration and OD values were determined using a NanoDrop 1000 microspectrophotometer, and RNA integrity was checked 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 cDNA template as in example 1.
5. Fluorescent quantitative PCR reaction
The long-chain RNA Lnc-13814 gene fluorescent quantitative detection primer nucleotide sequence is shown as SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
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
The SW 1.1 three-step method is used for carrying out the fluorescent quantitative PCR, and the amplification conditions are as follows: 10s at 98 ℃; 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 expression result of the long-chain RNA Lnc-13814 gene in the tissues of white follicles and yellow follicles is shown in a figure 3, and the result shows that, firstly, the expression of the long-chain RNA Lnc-13814 gene exists in the white follicles and the yellow follicles, and the long-chain RNA Lnc-13814 gene exists objectively in the follicular granulocytes of the laying duck. Secondly, the expression level of the long-chain RNA Lnc-13814 gene in the white follicle is remarkably higher than that in the yellow follicle (P < 0.01). Therefore, it can be seen that the long-chain RNA Lnc-13814 gene has an important correlation with the development of follicles.
Example 3 Effect of Long-chain RNA Lnc-13814 Gene overexpression on apoptosis of follicular granular cells of laying ducks
Construction of over-expression vector of long-chain RNA Lnc-13814 gene of egg-laying duck
The embodiment provides an egg-laying duck long-chain RNA Lnc-13814 gene overexpression vector, which comprises a cDNA sequence corresponding to the egg-laying duck long-chain RNA Lnc-13814. The vector construction specifically comprises the following steps:
1. double digestion treatment of pcDNA3.1(+) vector
The pcDNA3.1(+) vector is shown in FIG. 1, and contains restriction sites for KpnI and XhoI. The vector is subjected to double enzyme digestion by two endonucleases KpnI 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 as follows: the enzyme is cut for 6h at 37 ℃, and then the cut product is purified and recovered.
2. Double enzyme digestion of egg-laying duck long-chain RNA Lnc-13814 gene full-length fragment
The obtained target fragment, namely the egg-laying duck long-chain RNA Lnc-13814 gene PCR product is sequenced and verified, the correct PCR product verified in the example 1 is purified, the recovered PCR product is subjected to double enzyme digestion by two endonucleases KpnI and XhoI, the enzyme digestion reaction system is shown in the table 4, the total volume of the system is 50 mu L, and the reaction conditions are as follows: the enzyme is cut for 6h at 37 ℃, and then the cut product is purified and recovered.
TABLE 5
3. Construction of egg-laying duck long-chain RNA Lnc-13814 gene overexpression vector
Connecting the long-chain RNA Lnc-13814 gene fragment subjected to PCR amplification and double enzyme digestion with a vector subjected to double enzyme digestion, wherein a connection reaction system is as follows (the total volume is 10 mu L):
TABLE 6
The reaction conditions are as follows: ligation was performed overnight at 16 ℃.
Transforming the ligation product into competent cells, and performing colony PCR identification on positive clones, wherein the result is shown in FIG. 2, M in the figure represents DL2000DNA marker, and 1-2 in the figure represents colony PCR result of single colony; further, sequencing verification is carried out on the recombinant vector egg duck long-chain RNA Lnc-13814 gene overexpression vector, and the sequencing result is shown as SEQ ID NO: 1 is shown in the specification; the individual with correct colony PCR and sequencing result is the successfully constructed recombinant vector named 13814-pcDNA3.1.
Second, the separation of follicular granulosa 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 by using a scalpel (the action is quick), releasing yolk, and rinsing the residual yolk solution by using a PBS buffer solution to obtain the remaining follicle membrane: a basement membrane and a follicular granular 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.
Third, cell transfection
1. The long-chain RNA Lnc-13814 gene overexpression vector 13814-pcDNA3.1 and the empty vector pcDNA3.1(+) obtained in example 3 were diluted with 125. mu.L of Opti-MEM medium, respectively, and 5. mu. L P3000 was added thereto;
2. diluting 250 μ L of Opti-MEM medium
Fully and uniformly mixing the reagents;
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 as follows: firstly, transfecting pcDNA3.1(+) (empty vector group), secondly, transfecting 13814-pcDNA3.1 (long-chain RNA Lnc-13814 gene overexpression vector group). 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.
Four, fluorescent quantitative analysis
1. Extraction of Total RNA from cells
The cells were digested with Trizol, and 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 long-chain RNA Lnc-13814 gene fluorescent quantitative primer nucleotide sequence is shown as SEQ ID NO: 2 and SEQ ID NO: 3, the amplification conditions were: 10s at 98 ℃; 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 fluorescence quantitative primer of the marker gene BCL2 for inhibiting apoptosis is shown as SEQ ID NO: 6 and SEQ ID NO: 7, the amplification conditions were: 10s at 98 ℃; 94 ℃, 15s, 56 ℃, 15s, 60 ℃, 15s, 40 cycles; 1 cycle of 95 ℃ for 10s, 65 ℃ for 60s, 97 ℃ for 1 s;
(3) the nucleotide sequence of the fluorescent quantitative primer FAS of the apoptosis marker gene is shown as SEQ ID NO: 8 and SEQ ID NO: 9, the amplification conditions were: 10s at 98 ℃; 94 ℃, 15s, 54 ℃, 15s, 60 ℃, 15s, 40 cycles; 95 ℃ 10s, 65 ℃ 60s, 97 ℃ 1s, 1 cycle.
Fifth, apoptosis assay
1. Collecting a supernatant: the culture broth was collected in a flow tube (with a small amount of suspended cells).
2. Collecting cells: the cells in the six well plates were washed once with PBS, and 1ml of 0.25% pancreatin was added to digest the cells until the cells became round and some cells were suspended, i.e., PBS was added to stop the digestion. The cells were gently pipetted using a pipette gun to suspend the cells. Collected in a flow tube, centrifuged at 1500rpm for 5min and the supernatant discarded.
3. PBS-washed cells: 3ml of 4 ℃ pre-cooled PBS was added to completely resuspend the cells, and the cells were centrifuged at 1500rpm for 5min, and the supernatant was discarded. The pellet was resuspended in 300. mu.L of Binding Buffer.
4. Fluorescence labeling: add 5. mu.L Annexin V-FITC and mix, add 5. mu.L Propidium Iodide and mix. And reacting for 5-15min at room temperature in a dark place.
5. Testing the sample on the machine within 1 hour
Green fluorescence of Annexin V-FITC was detected by the FITC channel (FL1) and PI red fluorescence was detected by the PI channel (FL 2). Flow cytometer parameters were as follows: the excitation wavelength Ex is 488nm, and the emission wavelength Em is 530 nm.
Sixthly, result analysis
1. Quantitative fluorescence result analysis
Data were derived after the reaction was complete and results were used 2-ΔΔCTThe method is divided intoAnd (6) analyzing. Results of the analysis were plotted using Graphad Prism software. The analysis results of the expression quantity of the long-chain RNA Lnc-13814 gene and the expression quantity of the marker gene for inhibiting apoptosis and promoting apoptosis after transfection of the long-chain RNA Lnc-13814 overexpression vector of the laying duck are shown in the figure 4-5.
As can be seen from FIG. 4, after the long-chain RNA Lnc-13814 gene of the laying duck is over-expressed, compared with the empty vector group, the expression level of the long-chain RNA Lnc-13814 after the long-chain RNA Lnc-13814 gene of the laying duck is over-expressed is remarkably higher than that of the empty vector group (P < 0.01); therefore, the egg-laying duck long-chain RNA Lnc-13814 gene overexpression vector can stably express the egg-laying duck long-chain RNA Lnc-13814 gene.
As can be seen from FIG. 5, after the long-chain RNA Lnc-13814 gene of the laying duck is over-expressed, compared with the empty vector group, the expression level of the marker gene BCL2 for inhibiting apoptosis is greatly reduced (P <0.01), and the expression level of the marker gene FAS for apoptosis is greatly increased (P < 0.01). Therefore, the LncRNA can be used as a molecular marker for detecting the apoptosis condition in the follicular granular cells of the laying duck.
2. Apoptosis outcome analysis
The results of the flow cytometric assay are shown in FIG. 6. The results of the assay were analyzed and are shown in Table 7 below.
TABLE 7 statistical results of apoptosis detection by flow cytometry after overexpression of Long-chain RNA Lnc-13814
As can be seen from Table 7, after the long-chain RNA Lnc-13814 is over-expressed, the apoptosis number is increased, which shows that the long-chain RNA Lnc-13814 has a promoting effect on the apoptosis of the follicular granular cells of the laying duck, thereby further proving that the LncRNA can be used as a molecular marker for detecting the apoptosis condition of the follicular granular cells of the laying duck.
In conclusion, the egg-laying duck long-chain RNA Lnc-13814 gene overexpression vector can stably express the egg-laying duck long-chain RNA Lnc-13814 gene, can be used as a detection index to reflect the expression quantity of the egg-laying duck long-chain RNA Lnc-13814 gene, has the function of promoting the apoptosis of follicular granular cells, and can be applied to the detection of the apoptosis condition of the follicular granular cells of the egg-laying duck.
The egg-laying duck long-chain RNA Lnc-13814, the long-chain RNA Lnc-13814 and the long-chain RNA Lnc-13814 for regulating the follicular development of the egg-laying duck are all different names of the same RNA.
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
<120> long-chain RNA Lnc-13814 for regulating follicular development of laying duck and application thereof
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 974
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<213> egg-laying duck (cDNA)
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gtcagtgtgc tctgaggagc tgggggcttg ttcctgtggg ctcagagtac aaagtctgag 60
ccacttttga aagcaagaaa tggctgtcat taaggaaaaa cggcattcct gaggtcactt 120
cagagggagg tgtgattctc ccttgcagag cagaaccaag agcccaggaa ggtagcaagg 180
atgaagggaa tgagctgtgg ggacgggatt acctccagga gaggggctgg aggaatgcca 240
acagctaaat gaccgaacca ccttcaaagg aatttggaag atcgccttcc ccagccacca 300
ggctgctttg ggagatgcct ttcactgtgt tggggtgaat atcaccgagc aggaacagga 360
agaagactcc agcccttctg acacagcaga ggtttcctcc tagaagacag ggataaggag 420
ggcagagggc ttcatgtggg ccactgtggg actgtaacac cacgaaggcc aacgagaatt 480
gtgtgcaggg tggacaagga aacaaatacg tccgagagaa tcaacagttt gggaatatgc 540
aagttaactt tccatggctg tgtttctttg aaggtatccc cttcccctaa attttccttt 600
gctcctctca gttgtgtttc tgtccaaact ttcttcccct ccaaaagctg gaatactttc 660
attatattga aattaaatgg aaaaaaatat atatccccaa ctaaatctga aagtgatcaa 720
aaaatttaat acaaaaagcc atttttcctt gaaaacaatt cagctggctg ggatcaaatc 780
ttctcctgtt gaaacagcaa aattcccctt ggtttcagga accgtggggt gggtgcttgg 840
cagattttgg tgcccacaag ggcaacagac tcctaatttc atcagttgca gaactctttt 900
ccctgatcac cacatgggtt ttctgcctat ggattaggca catttggatt ttggagctcg 960
tctaccaggc acga 974
<210> 2
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<213> Artificial Sequence (Artificial Sequence)
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agcccaggaa ggtagca 17
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<213> Artificial Sequence (Artificial Sequence)
<400> 3
aaggcatctc ccaaagc 17
<210> 4
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cgtgtaattc tagttgttta aacacaaata cgtccgagag aatcaaca 48
<210> 5
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
caggtcgact ctagactcga gtcgtgcctg gtagacgagc t 41
<210> 6
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
acggctctcg ctcctgct 18
<210> 7
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
cggttgacgc tctccacg 18
<210> 8
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gccccagttg aagaaaaa 18
<210> 9
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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tccaggcaag taagaccc 18
Claims (7)
1. The application of the long-chain RNA Lnc-13814 detection reagent in preparing a detection kit for detecting the apoptosis condition of follicular granular cells of laying ducks is characterized in that the cDNA sequence corresponding to the long-chain RNA Lnc-13814 is shown as SEQ ID NO: 1 is shown.
2. The use of claim 1, wherein the long-chain RNA Lnc-13814 detection reagent comprises a fluorescent quantitative PCR detection primer pair comprising:
upstream primer 13814-F, as set forth in SEQ ID NO: 2;
downstream primer 13814-R, as set forth in SEQ ID NO: 3.
3. The use of claim 1, wherein the test kit comprises: the fluorescent quantitative PCR detection primer pair of claim 2.
4. The use of claim 3, wherein the detection kit 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, and the detection primer set according to claim 2.
5. The use of claim 4, wherein the fluorescent quantitative PCR reaction system is as follows: THUNDERBIRD SYBR qPCR Mix 10 uL, 10uM egg duck long-chain RNA Lnc-13814 upstream primer 0.4 uL, 10uM egg duck long-chain RNA Lnc-13814 downstream primer 0.4 uL, cDNA template to be detected 2.0 uL, ddH2O7.2 mu L, the total volume is 20 mu L;
wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 2 is shown in the specification;
the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 3, respectively.
6. The use of claim 3, wherein the method of using the test kit comprises the steps of:
step 1, extracting RNA in a sample to be detected, and then carrying out reverse transcription to obtain cDNA;
step 2, utilizing the primer pair in claim 2 to perform fluorescence quantitative PCR amplification on the cDNA of a sample to be detected, and adopting 2-ΔΔCTCalculating and obtaining the relative expression quantity of the long-chain RNA Lnc-13814 gene in the follicle sample from different sources by the method;
and 3, judging whether the egg-laying duck long-chain RNA Lnc-13814 gene and the expression condition of the egg-laying duck long-chain RNA Lnc-13814 gene exist in the sample to be detected according to the PCR amplification product and the relative gene expression quantity.
7. The use of claim 6, wherein the conditions of the fluorogenic quantitative PCR reaction in step 2 are, first denaturation: 10s at 98 ℃; and a second step of annealing: 94 ℃, 15s, 54 ℃, 15s, 60 ℃, 15s, 40 cycles; and a third step of extension: 95 ℃ for 10 s; 65 ℃, 60s, 97 ℃, 1s, 1 cycle.
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