CN109182562A - MiRNA apla-mir-25-42 relevant to laying duck follicular development and its detection primer, mortifier and application - Google Patents
MiRNA apla-mir-25-42 relevant to laying duck follicular development and its detection primer, mortifier and application Download PDFInfo
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- CN109182562A CN109182562A CN201811421003.7A CN201811421003A CN109182562A CN 109182562 A CN109182562 A CN 109182562A CN 201811421003 A CN201811421003 A CN 201811421003A CN 109182562 A CN109182562 A CN 109182562A
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention discloses a kind of miRNA apla-mir-25-42 relevant to laying duck follicular development and its detection primers, mortifier and application, belong to gene engineering technology field.The miRNA is apla-mir-25-42, and nucleotide sequence is as shown in SEQ ID NO:1.The studies have shown that of the invention miRNA can reduce the expression of cell proliferation marker gene C yclinB2, promote the expression of Apoptosis marker gene BCL2 by targeting the follicular development of TGFB2 gene regulation.The miRNA can be used as marker, for detecting laying duck follicular cell Proliferation, Differentiation situation；The existing vacancy in this field is compensated for, the Genetic Mechanisms of laying duck follicular development are provided fundamental basis and scientific basis, inheritance, raising laying duck reproductive capacity and genetic assistant breeding for studying laying duck reproductive trait all have great importance to parse.
Description
Technical field
The present invention relates to gene engineering technology fields, more particularly to miRNA apla- relevant to laying duck follicular development
Mir-25-42 and detection primer, mortifier and application.
Background technique
The egg laying performance of follicular development and laying duck is closely related, directly affects the egg production of laying duck.Existing research shows fowl
Class follicular development is extremely complex biological process, and people have centainly the follicular development mode of poultry at present
Solution, but as determine egg production an important factor for, the specific Regulation Mechanism of follicular development still needs to further investigate.
However currently, lack the miRNA marker of the follicular development situation of detection laying duck.
In consideration of it, the present invention is specifically proposed.
Summary of the invention
In order to overcome the drawbacks of the prior art with deficiency, the purpose of the present invention includes but is not limited to provide a kind of and laying duck ovum
It is soaked and educates relevant miRNA.The miRNA can be used as marker, for detecting laying duck follicular cell Proliferation, Differentiation situation,
To judge that the egg laying performance of target laying duck provides reference.
Another object of the present invention includes but is not limited to provide the application of above-mentioned miRNA.
Another object of the present invention includes but is not limited to provide the detection primer of above-mentioned miRNA.
Another object of the present invention includes but is not limited to provide a kind of suppression for inhibiting above-mentioned miRNA to be bound to its target gene
Object processed.
Another object of the present invention includes but is not limited to provide a kind of recombinant expression carrier.
Another object of the present invention includes but is not limited to provide a kind of method of regulation laying duck follicular development.
To achieve the above object, the present invention is achieved through the following technical solutions:
With the arriving of " genome era ", the entirety of character related gene expression mode is parsed in full-length genome level
Feature, with disclose laying duck lay eggs phenotypic character formation molecular basis be laying duck breeding work from now on important research direction it
One.MicroRNA (miRNA) by degradation said target mrna or can inhibit to turn over as a kind of small single-stranded endogenous non-coding RNA
Translate the regulating and controlling effect after playing its genetic transcription.In recent years, a large number of studies show that, miRNAs is in cell differentiation and apoptosis, growth
Crucial angle is play in multiple physiology such as development, glycolipid metabolism, inflammation, immune response and tumour generation and pathologic process
Color, wherein some miRNA for influencing follicular cell differentiation and being proliferated have been screened and have determined in Ovarian Follicles.
The upgrowth situation of granular cell directly influences the developmental state of ovarian follicle, and follicular cell can secrete sexual gland and swash
Growth, differentiation and the maturation of element and growth factor regulation thecacells and egg mother cell, while also accurately regulating and controlling ovarian follicle
Growth and development.Therefore, good follicular development is ensured, the regulation rule of grasp follicular development helps to further increase laying duck
Reproductive capacity is of great significance for improving laying duck production performance.
However at present about research especially laying duck ovarian follicle miRNA and its regulatory mechanism of laying duck tissue specificity miRNA
Research there is not been reported.Therefore, there is an urgent need to improve laying duck ovarian follicle miRNA research level and integrality, to be laying duck base
Because the research of group and its non-coding RNA provides the theoretical foundation of science, more to understand the regulation of laying duck follicular development in depth
Mechanism provides theory support to improve the reproductive performance of laying duck.
The present invention by carrying out high-throughput full transcript profiles sequencing to two groups of ovarian follicle tissue samples of different development stage, screening with
The relevant miRNA of laying duck follicular development is tentatively selected in significantly high table in white ovarian follicle by bioinformatics Differential expression analysis
The miRNA apla-mir-25-42 reached can promote cell proliferation marker by targeting the follicular development of TGFB2 gene regulation
The expression of gene C yclinB2 inhibits the expression of Apoptosis marker gene BCL2.The miRNA is to research laying duck reproductive trait
Inheritance and raising laying duck reproductive capacity have great importance, with important practical application value.
A kind of miRNA relevant to laying duck follicular development is provided in the first aspect of the present invention based on this, it is described
MiRNA is apla-mir-25-42, and nucleotide sequence is as shown in SEQ ID NO:1.
In the second aspect of the present invention, above-mentioned miRNA relevant to laying duck follicular development is provided as molecular marker
Application of the object in detection laying duck follicular cell Proliferation, Differentiation situation.
After study, expression quantity of the miRNA, that is, apla-mir-25-42 molecule provided by the invention in the white ovarian follicle of laying duck
The extremely significant expression quantity (P < 0.01) higher than in laying duck Huang ovarian follicle, finds laying duck apla-mir-25-42 and laying duck ovarian follicle for the first time
Development is related, and thus laying duck apla-mir-25-42 of the invention can be used as molecular marker for identifying the development of laying duck ovarian follicle
The Proliferation, Differentiation situation of situation such as follicular cell compensates for the existing vacancy in this field, to parse laying duck follicular development
Genetic Mechanisms provide fundamental basis and scientific basis；In addition, it is for studying the inheritance of laying duck reproductive trait, improving egg
Duck reproductive capacity and genetic assistant breeding all have great importance.
In the third aspect of the present invention, the primer for detecting above-mentioned miRNA is provided.
Further, in some embodiments of the present invention, the primer includes upstream shown in SEQ ID NO:2
LOOP primer shown in downstream primer shown in primer, SEQ ID NO:3 and SEQ ID NO:5.
In the fourth aspect of the present invention, a kind of above-mentioned miRNA knot relevant to laying duck follicular development of energy inhibition is provided
The mortifier of target gene is closed, the target gene is TGFB2 gene, and the binding site of the mortifier and the miRNA is
ATCCCCA；
Preferably, the mortifier is selected from 3 ' UTR of TGFB2 gene and is about 240bp section.
Further, in some embodiments of the present invention, the mortifier sequence is as shown in SEQ ID NO.4；
Preferably, restriction enzyme site sequence is added in the 5 ' ends and 3 ' ends of the mortifier respectively；
Preferably, the restriction enzyme site sequence generates cohesive end after digestion；
It is highly preferred that the restriction enzyme site sequence is respectively PmeI restriction enzyme site and XhoI restricted
The corresponding nucleotide sequence of restriction enzyme site；
It is highly preferred that the PmeI restriction enzyme site is located at 5 ' ends, the XhoI restriction enzyme site is located at 3 ' ends.
In the fifth aspect of the invention, a kind of recombinant expression carrier is provided, the recombinant expression carrier includes institute as above
The mortifier stated.
Further, in some embodiments of the present invention, the marker gene in the recombinant expression carrier is double glimmering
Light element enzyme reporter gene；
Preferably, the recombinant expression carrier is obtained by the way that the mortifier to be connected in report carrier pmirGLO
?.
In the sixth aspect of the present invention, a kind of reagent for detecting above-mentioned miRNA relevant to laying duck follicular development is provided
Box contains the primer sets for detecting above-mentioned miRNA.
Further, in some embodiments of the present invention, the primer sets include: reverse transcription primer, upstream primer
And downstream primer, reverse transcription primer sequence is as shown in SEQ ID NO:5；Upstream primer sequence is as shown in SEQ ID NO:2, downstream
Primer sequence is as shown in SEQ ID NO:3.
Using the primer sets, above-mentioned miRNA relevant to laying duck follicular development can be detected by stem-loop method.
Further, in some embodiments of the present invention, the kit contains for Reverse Transcription, reversion
Record primer, specific primer, internal control primer, quantitative fluorescent PCR reaction reagent.
The internal control primer includes the upstream primer and downstream primer of U6 reference gene, upstream primer sequence such as SEQ ID
Shown in NO:6, downstream primer sequence is as shown in SEQ ID NO:7.
As the preferred embodiment of kit of the present invention, the kit further includes that RNA extracts reagent, reverse transcription
Reaction system and quantitative fluorescent PCR reaction system, wherein the quantitative fluorescent PCR reaction system includes THUNDERBIRD
SYBR qPCR Mix、ddH2O, the cDNA and the detection primer pair that the reverse transcription reaction system reverse transcription obtains.
As the above specific embodiment, the quantitative fluorescent PCR reaction system is as follows: THUNDERBIRD SYBR
10 μ L, 10uM laying duck apla-mir-25-42 upstream primer of qPCR Mix, 0.4 downstream μ L, 10uM laying duck apla-mir-25-42
Primer 0.4 μ L, cDNA template 2.0 μ L, ddH to be detected27.2 μ L of O, 20 μ L of total volume.
In the seventh aspect of the present invention, the application method of the kit is provided, is included the following steps:
RNA in step 1, extraction sample to be tested；
Step 2 carries out apla-mir-25-42 reverse transcription generation using reverse transcription primer under the action of reverse transcriptase
cDNA；
Step 3 utilizes primer as described above (such as upstream primer shown in SEQ ID NO:2, SEQ ID NO:3 institute
LOOP primer shown in the downstream primer and SEQ ID NO:5 shown), fluorescent quantitative PCR is carried out to sample to be tested cDNA,
Using 2-ΔΔCTMethod calculates and obtains the relative expression quantity of apla-mir-25-42 in separate sources ovarian follicle sample；
Step 4 judges sample to be detected with the presence or absence of laying duck apla- according to pcr amplification product and gene relative expression quantity
Mir-25-42 and its expression.
As the above specific embodiment, the quantitative fluorescent PCR reaction condition in the step 3 is 98 DEG C, 10s；94
DEG C, 15s, 54 DEG C, 15s, 60 DEG C, 15s, 40 circulations；95 DEG C, 10s；65 DEG C, 60s, 97 DEG C, 1s, 1 circulations.
The eighth aspect of the present invention provides the analogies (i.e. mimic) of miRNA apla-mir-25-42, and sequence is such as
Shown in SEQ ID NO:1 and SEQ ID NO:8.
The ninth aspect of the present invention provides above-mentioned apla-mir-25-42 analogies in detection laying duck follicular cell
Application in Proliferation, Differentiation.
After study, inventors have found that the mimic of laying duck miRNA apla-mir-25-42 of the invention transfects entrance
In laying duck follicular cell, compared with NC group, the expression quantity of mimic group cell proliferation marker gene C yclinB2 is extremely significant
Increase (P < 0.01), the extremely significant reduction (P < 0.01) of the expression quantity of Apoptosis marker gene BCL2 shows that the miRNA can be used as
Molecular marker is applied to cell proliferation and differentiation situation in detection laying duck follicular cell.
In the tenth aspect of the present invention, the present invention provides a kind of methods of regulation laying duck follicular development comprising: to mesh
It marks laying duck follicular cell and applies reagent, the reagent contains as active constituent such as SEQ ID NO:1 and SEQ ID
RNA shown in NO:8.
Preferably, the regulation laying duck follicular development refers to: promoting laying duck follicular cell proliferation or inhibits laying duck ovum
Bubble granular cell tune is died.
Laying duck follicular cell is transfected using miRNA mimic shown in SEQ ID NO:1 and SEQ ID NO:8, is enabled
People surprisingly has found that the expression quantity of cell proliferation marker gene C yclinB2 increases, the expression of Apoptosis marker gene BCL2
Amount reduces, should be the result shows that miRNA shown in SEQ ID NO:1 can be used for regulating and controlling laying duck follicular development.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is relative quantification table of the laying duck apla-mir-25-42 provided by the invention in the white ovarian follicle of laying duck and yellow ovarian follicle
Up to result；
Fig. 2 be embodiment 2 in be overexpressed laying duck apla-mir-25-42 after, be overexpressed apla-mir-25-42 group with it is right
Situation of change is expressed according to apla-mir-25-42 in group；
Fig. 3 be embodiment 2 in be overexpressed laying duck apla-mir-25-42 after, be overexpressed apla-mir-25-42 group with it is right
Situation of change is expressed according to Cell apoptosis and proliferation key gene in group.
Fig. 4 is the structural schematic diagram of pmirGLO carrier.
Fig. 5 is the combination result of the 3 '-UTR of Dual-Luciferase method validation apla-mir-25-42 and TGFB2.
Specific embodiment
The acquisition PCR and its fluorescence quantitative detection kit and method of 1 laying duck miRNA apla-mir-25-42 of embodiment
One, laying duck apla-mir-25-42 is obtained
The corresponding nucleotide sequence of laying duck apla-mir-25-42 is as shown in SEQ ID NO:1 described in the present embodiment, this hair
Bright laying duck apla-mir-25-42 is applicant based on the full transcript profile sequencing result of laying duck ovarian follicle tissue completed early period, is passed through
One that a large amount of bioinformatic analysis screenings obtain is carried out to the sequencing result of two groups of ovarian follicle tissue samples of different development stage
New miRNA molecule.Applicant is named as laying duck apla-mir-25-42.
Two, the fluorescence quantitative PCR detection primer pair and its detection kit of laying duck miRNA apla-mir-25-42 and side
Method
The present embodiment design fluorescence quantitative PCR detection primer pair simultaneously identifies that the identification laying duck apla-mir-25-42 exists
Objective reality in laying duck follicular cell.The present embodiment identification identification method the following steps are included:
1, sample acquires
Acquire the white ovarian follicle of egg-laying peak laying duck and yellow each 3 parts of sample of ovarian follicle, PBS with syringe needle punctures ovarian follicle after cleaning up
Liquor folliculi is squeezed out, is put into the 1.5mL EP pipe of no enzyme, liquid nitrogen is put into after marking or -80 DEG C of refrigerators save.
2, sample Total RNAs extraction
Ovarian follicle total tissue RNA is extracted using traditional TRIzol, chloroform method, the specific steps are as follows:
1) liquid nitrogen grinding takes 50-100mg tissue sample to be put in mortar, and it is a little to pour into liquid nitrogen, grinds rapidly, to liquid nitrogen
Volatilization completely adds a small amount of liquid nitrogen, until tissue sample grinds as powder, tissue sample is transferred in 1.5mL centrifuge tube, is added
1mL Trizol tries temperature and places 5min, cracks it sufficiently；
2) 4 DEG C of 12000g are centrifuged 10min, supernatant are transferred in the 1.5mL centrifuge tube of clean no RNAase；
3) add 0.2ml chloroform according to every 1ml homogenate, violent 15s is placed at room temperature for 15min；
4) 4 DEG C of 12000g are centrifuged 10min；
5) upper layer colourless solution is carefully sucked out in centrifuge tube of another 1.5ml without RNAase, 0.5 times of isopropyl is added
Alcohol mixes, and tries temperature and stands 5-10min；
6) 4 DEG C of 12000g are centrifuged 10min, abandon supernatant, and RNA is sunken to tube bottom；
7) 1mL75% ethyl alcohol is added, mildly vibrates centrifuge tube, suspend precipitating；
8) 4 DEG C of 8000g are centrifuged 5min, abandon supernatant；
9) 1mL dehydrated alcohol is added, mildly vibrates centrifuge tube, suspend precipitating；
10) 4 DEG C of 8000g are centrifuged 5min, abandon supernatant, and room temperature is dried；
11) using 30-50 μ L without RNAase deionized water dissolving RNA, -80 DEG C are saved backup.
3, RNA sample quality testing is analyzed
It is solidifying using 1% agarose using 1000 micro-spectrophotometer of NanoDrop detection RNA sample concentration and OD value
The integrality of gel electrophoresis detection RNA
4, reverse transcription
Using the reverse transcription reagent box of Takara companyRT reagent Kit with gDNA
The specification step of Eraser carries out reverse transcription and obtains cDNA template, and wherein reverse transcription primer sequence is apla-mir-25-42-
LOOP (as shown in SEQ ID NO:5).
1) genomic DNA remove dereaction
Table 1
Reaction condition is as follows:
42℃2min；4 DEG C of preservations.
2) reverse transcription reaction
Reaction solution process for preparation carries out on ice.
Table 2
Reaction condition is as follows:
37℃,15min；85℃,5sec；4 DEG C of preservations.
5, quantitative fluorescent PCR reacts
Apla-mir-25-42 fluorogenic quantitative detection primer nucleotide sequences such as SEQ ID NO:2 and SEQ ID NO:3 institute
Show, the primer nucleotide sequences for reference gene fluorogenic quantitative detection are as shown in SEQ ID NO:6 and SEQ ID NO:7.
Using Takara company Real time kit THUNDERBIRD SYBR qPCR Mix specification step into
Row quantitative fluorescent PCR, reaction system are following (20 μ L):
Table 3
Utilize Roche1.1 three-step approach of 96SW carries out quantitative fluorescent PCR, amplification condition are as follows: and 98 DEG C,
10s；94 DEG C, 15s, 54 DEG C, 15s, 60 DEG C, 15s, 40 circulations；95 DEG C of 10s, 65 DEG C of 60s, 97 DEG C of 1s, 1 circulation.
Data are exported after reaction, as a result utilize 2-ΔΔCTMethod is analyzed.Utilize Graphad Prism software pair
Analysis result is mapped.
Apla-mir-25-42 expression of results is shown in Fig. 1 in white ovarian follicle and yellow ovarian follicle tissue, the results show that first, white ovarian follicle
In and yellow ovarian follicle in have the expression of apla-mir-25-42, the laying duck apla-mir-25-42 is thin in laying duck granulosa
Objective reality in born of the same parents.Second, in white ovarian follicle the expression quantity of apla-mir-25-42 it is extremely significant be higher than in yellow ovarian follicle (P <
0.01).Thus, it will be seen that the development of apla-mir-25-42 and ovarian follicle have important correlation.
Embodiment 2apla-mir-25-42 is as molecular marker in detection laying duck follicular cell Proliferation, Differentiation
Using
One, laying duck follicular cell separates
The separation of laying duck follicular cell uses following steps:
1, the laying ducks of egg-laying peak, jugular vein sacrificed by exsanguination are selected；Entire ovary tissue is taken out, is placed in equipped with pre-cooling
In the sterile petri dish of PBS；
2, blood stains are rinsed with added with dual anti-PBS buffer solution, rinsed 3 times；
3, the ovarian follicle of rinsed clean is moved into the plate equipped with pre-cooling PBS buffer solution, peels theca of follicle of von Baer, connective tissue off
And rete vasculosum；
4, yolk is discharged, is buffered with PBS in the notch (movement is fast) of the standardized road 1-2cm long in ovarian follicle surface with scalpel
Remaining yolk liquid rinsed clean, remaining membrana follicularis be by liquid are as follows: basement membrane and follicular cell layer；
5, the membrana follicularis of rinsed clean is shredded as far as possible, is placed in 15mL centrifuge tube, add 4mL culture medium, with 1mL liquid-transfering gun
1min is blown and beaten repeatedly, abandons supernatant after 4 DEG C of 1000rpm centrifugations；
6,0.2% II Collagenase Type of 4mL is added into precipitating, precipitating is resuspended, is placed in 37 DEG C of constant-temperature table 80rpm digestion
30min；
7, digestion terminates, and 4mL M199 complete medium (containing 10% serum) is added and terminates digestion；It is sieved with 200 mesh stainless steels
Filtering, and mesh screen is cleaned with 2mL M199 complete medium, filtrate is collected, 4 DEG C of 1000rpm are centrifuged 10min；
8, supernatant is abandoned, 10mL M199 complete medium is added, 4 DEG C of 1000rpm are centrifuged 10min；
9, supernatant is abandoned, after M199 complete medium (dual anti-containing 10%FBS and 1%) resuspension is added, cell density is measured, adjusts
Whole suspension cell density is 1 × 106A/mL is inoculated in 6 hole culture dishes, is placed in 37 DEG C, 5%CO2Stationary culture in incubator；
10, after granulosa cell culture 24 hours, renew the fresh M199 culture medium containing fetal calf serum, attached cell can be used for
Further in research.
Two, cell transfecting
1, apla-mir-25-42mimic (the i.e. SEQ ID of 125 μ L Opti-MEM culture mediums dilution synthesis is taken respectively
NO:1 and SEQ ID NO:8) and NC；
2,250 μ L Opti-MEM culture mediums is taken to dilute3000 reagents, mix well；
3, the mixture in 125 μ L (2) is added in two pipe mixtures in (1) respectively, is incubated at room temperature 5min；
4,250 μ L mixtures are transferred to cell.
3 repeating holes are arranged in each experimental group, and transfection cell is grouped as follows: 1. being transfected NC, is 2. transfected apla-mir-25-42.
After each transfection group transfects cell for 24 hours, using Trizol vitellophag, fluorescent quantitation after overexpression is used for after extracting cell total rna
Analysis.
Three, quantitative fluorescence analysis
1, in cell total serum IgE extraction
Total serum IgE in cell is extracted referring to the extracting method of total serum IgE in embodiment 1.
2, reverse transcription
With the method for reverse transcription in embodiment 1.
3, quantitative fluorescent PCR reacts
(1) quantitative fluorescent PCR reaction condition and method be with embodiment 1, wherein apla-mir-25-42 fluorescent quantitation primer
Nucleotide sequence is as shown in SEQ ID NO:2 and SEQ ID NO:3, amplification condition are as follows: and 98 DEG C, 10s；94 DEG C, 15s, 54 DEG C,
15s, 60 DEG C, 15s, 40 circulations；95 DEG C of 10 s, 65 DEG C of 60s, 97 DEG C of 1s, 1 circulation；
(2) cell Proliferation marker gene CyclinB2 fluorescent quantitation primer nucleotide sequences such as SEQ ID NO:9 and SEQ
Shown in ID NO:10, amplification condition are as follows: 98 DEG C, 10s；94 DEG C, 15s, 56 DEG C, 15s, 60 DEG C, 15s, 40 circulations；95℃
10s, 65 DEG C of 60s, 97 DEG C of 1s, 1 circulation；
(3) Apoptosis marker gene BCL2 fluorescent quantitation primer nucleotide sequences such as SEQ ID NO:11 and SEQ ID
Shown in NO:12, amplification condition are as follows: 98 DEG C, 10s；94 DEG C, 15s, 54 DEG C, 15s, 60 DEG C, 15s, 40 circulations；95 DEG C of 10s, 65
DEG C 60s, 97 DEG C of 1s, 1 circulation；
(4) reference gene fluorescent quantitation primer nucleotide sequences are as shown in SEQ ID NO:6 and SEQ ID NO:7, amplification
Condition are as follows: 98 DEG C, 10s；94 DEG C, 15s, 54 DEG C, 15s, 60 DEG C, 15 s, 40 circulations；95 DEG C of 10s, 65 DEG C of 60s, 97 DEG C of 1s, 1
A circulation.
4, interpretation of result
Data are exported after reaction, as a result utilize 2-ΔΔCTMethod is analyzed.Utilize Graphad Prism software pair
Analysis result is mapped.It the expression quantity of apla-mir-25-42 and cell Proliferation, withers after transfection laying duck apla-mir-25-42
The expression analysis result for dying marker gene is shown in Fig. 2-3.
As seen from Figure 2, after being overexpressed apla-mir-25-42, compared with NC group, it is overexpressed laying duck apla-mir-
The expression quantity of apla-mir-25-42 is extremely significant after 25-42 is higher than NC group (P < 0.01).
As seen from Figure 3, after being overexpressed laying duck apla-mir-25-42, compared with NC group, it is overexpressed laying duck apla-
After mir-25-42, the extremely significant increase (P < 0.01) of the expression quantity of cell proliferation marker gene C yclinB2, Apoptosis mark
The extremely significant reduction (P < 0.01) of the expression quantity of gene BCL2, illustrates that the miRNA promotes the proliferation of cell.It can be seen that should
MiRNA can be used as molecular marker and be applied to cell proliferation and differentiation situation in detection laying duck follicular cell.
In conclusion the laying duck apla-mir-25-42mimic of the synthesis in the present invention can stablize expression laying duck apla-
Mir-25-42, and can be used as the expression quantity of Testing index reflection laying duck apla-mir-25-42, and can be applied to detection egg
Cell proliferation and differentiation situation in duck follicular cell.
The design of 3 laying duck apla-mir-25-42 target gene TGFB2 luciferase reporter gene detection system of embodiment
With building
Firstly, it is associated analysis in full transcript profile sequencing result for selected miRNA apla-mir-25-42,
The target gene TGFB2 with apla-mir-25-42 interaction is filtered out from the sequencing result of mRNA, is analyzed using miRBase
The binding site for obtaining apla-mir-25-42 and TGFB2 is ATCCCCA.
Then, the 3 '-UTR sequences (i.e. the mortifier of apla-mir-25-42) that TGFB2 is obtained from NCBI, utilize mutation
ATCCCCA in the mortifier of apla-mir-25-42 is sported CAGGCGC by method, respectively by above-mentioned two genetic fragment
It is cloned into pmirGLO carrier, obtains the carrier (pmirGLO- of the mortifier sequence containing apla-mir-25-42 accordingly
TGFB2-wt the carrier (pmirGLO-TGFB2-mut) of the mortifier mutant nucleotide sequence) and containing apla-mir-25-42.Carrier
Building specifically comprises the following steps:
(1) the double digestion processing of pmirGLO carrier
The structural schematic diagram of pmirGLO carrier is as shown in figure 4, carrier contains restriction enzyme PmeI and XhoI digestion
Site.Double digestion is carried out to above-mentioned carrier with two kinds of restriction endonuclease PmeI and XhoI, endonuclease reaction system is as follows, and system total volume is
50μL:
Table 4
Reaction condition are as follows: 37 DEG C of digestion 6h then carry out purification and recovery to digestion products.
(2) acquisition and verifying of 3 ' the UTR segment of laying duck TGFB2 gene of mutation front and back
For the sequence in 3 ' UTR of laying duck TGFB2 gene with the binding site of apla-mir-25-42 mutation front and back, respectively
3 ' the UTR segment of TGFB2 gene of design primer amplification mutation front and back, the amplimer nucleotide sequence point before and after the mutation of use
Not as shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, following (20 μ of reaction system
L)。
Table 5
Amplification condition are as follows: 95 DEG C, 300s；94 DEG C, 30s, 56 DEG C, 30s, 72 DEG C, 30s, 30 circulations；72 DEG C, 600s.
The PCR product that amplification obtains is detected using 1% agarose gel electrophoresis and send sequence verification.
(3) double digestion of 3 ' the UTR segment of laying duck TGFB2 gene of mutation front and back
It is purified to correct PCR product is verified, with two kinds of restriction endonuclease PmeI and XhoI to the above-mentioned PCR product of recycling
Double digestion is carried out, endonuclease reaction system is as follows, and the total volume of system is 50 μ L:
Table 6
Reaction condition are as follows: 37 DEG C of digestion 6h then carry out purification and recovery to digestion products.
(3) building of pmirGLO-TGFB2-wt and pmirGLO-TGFB2-mut carrier
3 ' UTR segment of TGFB2 gene by PCR amplification and after double digestion connects with the carrier after double digestion is also passed through
It connects, coupled reaction system is following (10 μ L of total volume):
Table 7
Reaction condition are as follows: 16 DEG C of connections overnight.
Connection product is transformed into competent cell, bacterium colony PCR identification and sequence verification, sequencing knot are carried out to positive colony
Fruit is as shown in SEQ ID NO:4 and SEQ ID NO:17；The recombinant vector that success constructs, is respectively designated as pmirGLO-TGFB2-
Wt and pmirGLO-TGFB2-mut.
The mechanism of 4 apla-mir-25-42 of embodiment promotion laying duck follicular cell apoptosis
Laying duck follicular cell is separated, according to 5 × 104/ hole is seeded in 24 orifice plates, with pmirGLO, pmirGLO-
TGFB2-wt, pmirGLO-TGFB2-mut respectively with apla-mir-25-42mimic cotransfection.It is collected afterwards for 24 hours in cotransfection thin
Born of the same parents utilizeReporter Assay System detects the relative fluorescence of fluorescence carrier in microplate reader
Activity, testing result are as shown in Figure 5.It is and prominent the result shows that after cotransfection apla-mir-25-42 and TGFB2 wild type carrier
Modification and empty carrier corotation group are compared, and Dual-Luciferase activity significantly reduces (P < 0.05), illustrate apla-mir-25-42
Mimic can in conjunction with 3 ' UTR of TGFB2 gene, this result illustrate apla-mir-25-42 can by targeting combine TGFB2
3 ' UTR of gene check TGFB2 expression, to promote the proliferation of laying duck follicular cell.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
SEQUENCE LISTING
<110>Animal Husbandry and Veterinary Inst., Hubei Academy of Agricultural Sciences
<120>miRNA apla-mir-25-42 relevant to laying duck follicular development and its detection primer, mortifier and application
<160> 17
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> RNA
<213>artificial sequence
<400> 1
guggggauuu guugcauuac uu 22
<210> 2
<211> 32
<212> DNA
<213>artificial sequence
<400> 2
acactccagc tggggtgggg atttgttgca tt 32
<210> 3
<211> 16
<212> DNA
<213>artificial sequence
<400> 3
tggtgtcgtg gagtcg 16
<210> 4
<211> 240
<212> DNA
<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 (10)
1. a kind of miRNA relevant to laying duck follicular development, which is characterized in that the miRNA is apla-mir-25-42, core
Nucleotide sequence is as shown in SEQ ID NO:1.
2. miRNA relevant to laying duck follicular development is as molecular marker in detection laying duck ovarian follicle as described in claim 1
Application in granulosa cell proliferation differentiation situation.
3. detecting the primer of miRNA marker relevant to laying duck follicular development as described in claim 1.
4. primer according to claim 3, which is characterized in that the primer includes that upstream shown in SEQ ID NO:2 is drawn
LOOP primer shown in downstream primer shown in object, SEQ ID NO:3 and SEQ ID NO:5.
5. a kind of mortifier for inhibiting target gene in conjunction with miRNA relevant to laying duck follicular development described in claim 1, special
Sign is, the target gene is TGFB2 gene, and the binding site of the mortifier and the miRNA are ATCCCCA；
Preferably, the mortifier is selected from 3 ' UTR of TGFB2 gene and is about 240bp section.
6. mortifier as claimed in claim 5, which is characterized in that the nucleotide sequence of the mortifier such as SEQ ID NO.4
It is shown；Preferably, restriction enzyme site sequence is added in the 5 ' ends and 3 ' ends of the mortifier respectively；Preferably, described
Restriction enzyme site sequence generates cohesive end after digestion；It is highly preferred that the restriction enzyme site sequence is distinguished
For PmeI restriction enzyme site and the corresponding nucleotide sequence of XhoI restriction enzyme site；It is highly preferred that the PmeI enzyme
Enzyme site is located at 5 ' ends, and the XhoI restriction enzyme site is located at 3 ' ends.
7. a kind of recombinant expression carrier, which is characterized in that the recombinant expression carrier includes as described in claim any one of 5-6
Mortifier.
8. recombinant expression carrier as claimed in claim 7, which is characterized in that the marker gene in the recombinant expression carrier is
Luciferase reporter gene；
Preferably, the recombinant expression carrier is obtained by the way that the mortifier to be connected in report carrier pmirGLO.
9. miRNA as described in claim 1 or such as described in any item mortifiers of claim 5-6 are thin in laying duck granulosa
Application in born of the same parents' Proliferation, Differentiation.
10. a kind of method of regulation laying duck follicular development, characterized in that it comprises: applied to target laying duck follicular cell
With reagent, the reagent contains the RNA as shown in SEQ ID NO:1 and SEQ ID NO:8 as active constituent；
Preferably, the regulation laying duck follicular development refers to: promoting laying duck follicular cell proliferation or inhibits laying duck ovarian follicle
Granulocyte tune is died.
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| CN110923233A (en) * | 2019-12-16 | 2020-03-27 | 华南农业大学 | miRNA influencing development of goat follicle, and screening method and application thereof |
| CN114517198A (en) * | 2022-03-09 | 2022-05-20 | 中国水产科学研究院黄海水产研究所 | Cynoglossus semilaevis miRNA and application thereof in regulating and controlling tgfb2 gene expression |
| CN114517198B (en) * | 2022-03-09 | 2023-06-16 | 中国水产科学研究院黄海水产研究所 | Cynoglossus semilaevis miRNA and application thereof in regulation and control of tgfb2 gene expression |
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