CN107974462A - It is a kind of to improve the method for lean pork yield by postponing the expression time of gene - Google Patents
It is a kind of to improve the method for lean pork yield by postponing the expression time of gene Download PDFInfo
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- CN107974462A CN107974462A CN201710295934.6A CN201710295934A CN107974462A CN 107974462 A CN107974462 A CN 107974462A CN 201710295934 A CN201710295934 A CN 201710295934A CN 107974462 A CN107974462 A CN 107974462A
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- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
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Abstract
The invention discloses a kind of the method for lean pork yield is improved by postponing the expression time of gene, specifically by Pig embryos early stage is postponed gene M yoD expression times are determined into flesh, postponed so that embryo's early stage primary muscle fibre forms the time, increase muscle fibre sum, so as to improve meat yield after birth（Lean meat yield）.Present invention research first is found, the more pig myoD expression of meat yield is later, and the appearance primary muscle fibre time is also later, therefore sarcoblast has more times to breed, the muscle fibre quantity that is produced after the differentiation pig kind fewer than meat yield is more, and finally meat yield is also just more after birth.Based on this important discovery, the present invention can influence the expression time of even MyoD in change animal body by animal nutrition, so as to obtain the livestock and poultry new varieties more than meat yield.
Description
Technical field
The present invention relates to molecular genetic techniques field, and in particular, to a kind of to be carried by postponing the expression time of gene
The method of high lean pork yield.
Background technology
The skeletal development of pig mainly has two-wheeled in embryonic period, embryonic phase：1）The formation stages of primary muscle fibre, gestation after the 14th~
It is within 22 days the development of body segment, subsequent paraxial mesoderm develops to form myotome, and primary muscle fibre is opened for the 35th day or so after gestation
Begin to occur, reach peak of proliferation within the 49th day or so in gestation, and be continued until gestation the 60th day.2）Secondary muscle fibre forms rank
Section, the secondary muscle fibre of gestation the 45th ~ 90 day or so surrounds differentiation and development around primary muscle fibre, and reaches within 75 days or so in gestation
Peak, gestation is after the 91st day, and muscle fibre number no longer changes substantially, and muscle fibre is substantially carried out the hypertrophy of muscle fibre after birth
And the maturation of Change of types.Therefore, the muscle fibre sum of Pig embryos phase skeletal muscle just has determined before birth.
Forefathers study discovery, and muscle fibre number and size are the main determining factor of meat yield, among these, muscle fibre number
It is more important, because the higher animal of the Muscle fiber density being of moderate size can produce more lean meat and preferable meat, and
Muscle fibre number is more, and the speed of animal individual growth is more fast more efficient.First filial generation is not after bigger white and Landrace hybridizes
With the embryo of size, it is found that time that primary muscle fibre occurs does not have difference, but small Pig embryos secondary muscle fibre
Quantity will be big considerably less than build embryo, and have the difference of 17% muscle fibre sum after birth.Therefore in same kind
Interior, secondary muscle fibre may be to the influence bigger of total muscle fibre number.Comparison between different cultivars pig, the larger great Bai of build
The number of pig primary muscle fibre and the ratio of secondary muscle fibre and primary muscle fibre are all significantly higher than miniature pig.Therefore, not
With between build pig variety, primary muscle fibre may be to the influence bigger of total muscle fibre number.In conclusion primary muscle fibre is producing
In the different pig kind of meat amount, play the role of to postnatal meat yield conclusive.
It is into the gene family to play a decisive role during flesh into flesh regulatory factor family.There are four members in the family：
MyoD, Myf5, Myogenin and MRF4, its common trait are to possess one to have into the highly conserved of flesh regulation and control potentiality
Basic helix-loop-helix structure function domain.Among four members, Myf5 is expressed at first in embryo, and is complementary to one another with MyoD
Gene is determined as the starting into flesh.Studies have found that the transgenic mice of missing above-mentioned two gene will not form any flesh
Meat, but above-mentioned any one gene in both is individually lacked, to not having too much influence into flesh, illustrate Myf5 and MyoD
There is certain function overlapping and complementary.Research discovery is being carried out to knocking out separated sarcoblast in MyoD mouse, is knocking out MyoD
Sarcoblast can breed the longer time, cause differentiation delay.Nearest result of study shows that MyoD is combined down with Myf5
It is almost consistent to swim the site of gene, but compared with MyoD, Myf5 lacks a stronger activation domain, therefore without MyoD that
The strong ability for causing downstream gene expression.
Zhao Xiao etc. arrives birth in 35 days in 2011, using Guangdong local pig breed indigo plant pool pig and external lean meat species Landrace embryo
The longissimus dorsi muscle tissue of 180 days has carried out morphologic observation and transcript profile sequencing afterwards.It turns out that blue pool pig in embryo 35 days just
Through there is primary muscle fibre, and Landrace has arrived the talent of embryo 49 and primary muscle fibre has occurred, but in muscle development afterwards
During, the development of blue pool pig muscle will be slower than Landrace, and after being born meat yield also below the latter.It is sequenced by transcript profile,
They have found more to express earlier than Landrace in blue pool Pig embryos early stage into flesh factor of determination, just include MyoD among these
Gene.
2015, local pig breed Tongcheng pig and the west of another Central China of Yuqiang Zhao et al. comparative studies
Square Fastback Yorkshire（Large White）The longissimus dorsi muscle tissue of 5 weeks after birth of embryo 30 days.With blue pool pig situation class
Seemingly, tectology find, the less Tongcheng pig of meat yield equally 30 days embryonic period, embryonic phases Bjork summer pig possess a greater number and
The sarcoblast of higher density, but in the second wheel muscle development afterwards, the development speed of the secondary muscle fibre of Yorkshire
Degree will be faster than Tongcheng pig.
The quantity of Pig embryos beginning level muscle fibre determine muscle fibre sum, and muscle fibre sum can influence it is postnatal
Meat yield, at the same muscle fibre sum in embryonic period, embryonic phase it has been determined that being produced after therefore the development of Pig embryos beginning level muscle fibre is to birth
Meat measurer has decisive influence.
The content of the invention
The purpose of the invention is to overcome the above-mentioned deficiency of the prior art, there is provided it is a kind of by the expression that postpones gene when
Between improve the method for lean pork yield.
To achieve these goals, the present invention is achieved by the following technical programs：
Postpone application of the expression time of Pig embryos phase myogenic differentiation factor M yoD in pig meat yield is improved.
It is a kind of to improve the method for lean pork yield by postponing the expression time of gene, postpone Pig embryos phase myogenic differentiation
The expression time of factor M yoD, so as to improve pig meat yield.Preferably, MyoD genes are postponed and are expressed in pig 1 week and are increased with reaching
Add the effect of meat yield.
The present invention simulates MyoD genes in Chinese native pig breed in the C2C12 sarcoblasts system in mouse source and early expresses
Situation, MyoD gene expressions 48h and 24h in advance, reduces the myotube quantity that differentiation produces respectively, and more early expression production
Raw myotube quantity is fewer.In practical applications, we are according to the expression more late than miniature pig Wuzhi Mountain pig MyoD of lean meat species Landrace
The actual result of one week, postpones MyoD genes and 1 week is expressed in pig to reach the effect of increase meat yield.
Length that the present invention is successively decreased using meat yield successively is white, the blue pool and Wuzhi Mountain Pig embryos 18,21,28,32,35 and of early stage
The longissimus dorsi muscle meat sample tissue of 42 days carries out Histomorphological and the detection of myotube marker protein, it has been found that three breeding pigs
The time that primary muscle fibre occurs has differences：Wuzhi Mountain pig occurs primary muscle fibre for 32 days first in embryo, and Lan Tang and length are white
Pig occurs primary muscle fibre for 35 days in embryo, and the primary muscle fibre quantity of blue pool pig is significantly higher than Landrace at this time.By right
Above-mentioned sample carries out transcript profile sequencing, quantitative PCR and Western blot analyses, it has been found that MyoD meat yield it is minimum five
Refer to and earliest, highest is expressed in the pig of mountain（Embryo starts to express for 21 days）, and expressed in the most Landrace of meat yield the latest, it is minimum
（Embryo starts to express for 28 days）.In order to verify myoD expression times to the influence into flesh, we are in C2C12 and NIH3T3 cell lines
In by being overexpressed MyoD plasmids verify the expression of MyoD and whether expression time can influence the muscle fibre number that finally breaks up
Amount, and result complies fully with expection, i.e., MyoD is overexpressed in two kinds of cell line can suppress the propagation of cell, be produced after differentiation
Myotube reduce.Based on this important discovery, we can influence even to change in animal body by animal nutrition
The expression time of MyoD, so as to obtain the livestock and poultry new varieties more than meat yield.Realize that the method for postponing expression MyoD has in Native Pig
The technology of MyoD genes is knocked in kind using the dox Cre-LoxP induced.Pass through Cre-LoxP pigs and CRISPR-Cas9 technology structures
The MyoD built knocks in the MyoD-LacZ-Stop-Cas9-KI pigs that pig hybridizes, in different embryonic stages to sow feeding
Dox, induces the sequential of MyoD gene expressions, postpones local pig breed MyoD gene expression times.In addition with utilizing slow virus
The RNA perturbation techniques of mediation, that is, build the slow virus interference carrier of MyoD genes, the mother in different embryonic stages to local pig breed
Pig injecting lentivirus interference carrier is so as to postpone the expression time of MyoD.The result of these methods is exactly sarcoblast is had more
Time propagation, and then differentiate more muscle fibres, improve the lean meat yield of local pig breed.
Compared with prior art, the present invention has the advantages that：
Present invention firstly discovers that：The more pig MyoD expression of meat yield is later, and the appearance primary muscle fibre time is also later, because
This sarcoblast has more times to breed, and the muscle fibre quantity produced after differentiation the pig kind fewer than meat yield is more, is finally going out
Meat yield is also just more after life.Based on this important discovery, the present invention can influence even to change by animal nutrition
Become the expression time of MyoD in animal body, so as to obtain the livestock and poultry new varieties more than meat yield.
Brief description of the drawings
Fig. 1 is the experimental verification figure that three breeding pig embryos early stage primary muscle fibre time of occurrence has differences；A.HE is dyed,
400×；B. immunohistochemistry, 400 ×；C. the quantity statistics of primary muscle fibre；D. three breeding pig embryo's longissimus dorsi muscle histogenic immunity
Western blot result；Immunohistochemistry, WB experimental antibodies are MYHC, and internal reference GAPDH, signified arrow is primary muscle fibre；Scale=
50μm；*, p<0.05；*, p<0.01.
Fig. 2 is that three breeding pig embryos influences the transcript profile sequencing result into oligogene/miRNA of flesh in five periods.（A-
B）MRFs and MEFs family genes；（C）The marker gene in myogenic differentiation period；（D）Suppress into the gene of flesh；（E）Promote into flesh
Into the special miRNA of flesh.
Fig. 3 is the experimental verification figure that three breeding pig embryos early stage primary muscle fibre time of occurrence has differences.
Fig. 4 is to be overexpressed MyoD gene efficiency proof diagrams.A. MyoD genes are overexpressed, it is thin to dye detection C2C12 using EDU
Born of the same parents breed.B. just the ratio of total cell is accounted in proliferative cell.C. it is overexpressed the influence of MyoD gene cell cycles.D. it is overexpressed
After MyoD genes, C2C12 cell Proliferations are detected using real-time n cell detecting system.E. after being overexpressed MyoD genes, Q-
PCR quantitatively detects the expression of cell cycle correlation factor gene.
Fig. 5 is the influence that different time points are overexpressed MyoD gene pairs myogenic differentiations.A. 2 days before breaking up（-2d）And differentiation
First 1 day（-1d）MyoD genes are overexpressed, pCMV empty plasmids are negative control.Differentiation, Western-blot detections are induced at the same time
The expression of MYHC albumen.B. different time points are overexpressed MyoD genes, myotube immunofluorescence results figure.C. for figure A's
Western-blot detects the gray scale scanning quantitative result of MYHC albumen.
Fig. 6 is to be overexpressed the figure that MyoD makes NIH3T3 cell fusions form myotube.（A）After being overexpressed MyoD, MyoD is detected
MRNA expressions.（B）After transfecting 48h, MyoD protein levels are detected.（C）After induction differentiation 7d, myogenic differentiation key factor
MRNA level in-site detection.（D）After induction differentiation 7d, observe that myotube is formed under white light.（E）After being overexpressed MyoD, induction differentiation
7d, the expression of Immunofluorescence test to MyHC.（F）After being overexpressed MyoD, induction differentiation 7d, protein level detects the table of MyHC
Reach..*p<0.05, * * p<0.01, * * * p<0.001, n=3.Scale size：100μm.
Fig. 7 is to be overexpressed MyoD, suppresses the figure of the propagation of NIH3T3 cells.（A）Real-time n cell detecting system inspection
Survey after being overexpressed MyoD, the proliferative conditions of NIH3T3 cells compared with control group.（B）Count GM different times control group and cross table
Up to the cell number of group.（C）Be overexpressed MyoD after, PI dyeing, the flow cytometry cell cycle, be overexpressed MyoD after with it is right
Compared according to group, the NIH3T3 cells generation G1 phases block, S phase Leukopenias.（D）Immunofluorescence test EdU positive cells simultaneously count.
（E）The mRNA level in-site detection of cell cycle key factor.*p<0.05, * * p<0.01, * * * p<0.001, n=3.Scale size：
100μm。
Embodiment
The present invention is made with reference to Figure of description and specific embodiment and further being elaborated, the embodiment
It is served only for explaining the present invention, is not intended to limit the scope of the present invention.Test method used in following embodiments is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
Embodiment 1 grows determining for white, the blue pool and the Wuzhi Mountain pig primary muscle fibre time of occurrence
1st, sample collection：Long white, the blue pool and Wuzhi Mountain pig select 12 mothers of full sibs or half sibs all in accordance with respective pedigree
Pig, respectively carries out artificial insemination using the sperm of the corresponding kind boar of same head（Wuzhi Mountain pig uses nature insemination method）, in phase
With rearing conditions under the identical feed of feeding.The treating method of all animals is all in strict accordance with Guangdong Province's animal protection with using
The flow of committee's approval carries out, and authentication code is：SCXK (Guangdong) 2011-0029 and SYXK (Guangdong)
2011–0112.Butcher each 2 sows of kind respectively embryonic period, embryonic phase 18,21,28,32,35 and 42 days, cut open the belly and cut off uterus taking-up
Embryo.Due to the smaller event rounding embryo of embryo, remaining period separation embryo back meat sample tissue, is cut into about soya bean within 18 and 21 days
The tissue block of size.All samples use PBS wash cleans, and a part is fitted into 2.0 mL cryopreservation tubes, and is put into liquid nitrogen rapidly
Preserve；It is spare that another part is put into 4% paraformaldehyde solution.For the ease of mark, three breeding pigs are abbreviated as respectively：Landrace
（LR）, blue pool pig（LT）With Wuzhi Mountain pig（WZS）；6 embryonic stages are abbreviated as respectively：18 days (LR1/LT1/WZS1), 21 days
（LR2/LT2/WZS2）, 28 days（LR3/LT3/WZS3）, 32 days（LR4/LT4/WZS4）, 35 days（LR5/LT5/WZS）With 42 days
（LR6/LT6/WZS6）.
2nd, paraffin section and HE dyeing：(1) it is fixed：When 4% paraformaldehyde placement 24~48 is small；(2) it is dehydrated：Sample is put
Enter in embedded box, carry out mark, be then placed in fully-automatic dewatering machine, dehydration is：70% ethanol：2h；80% ethanol：2h；
95% ethanol：30min；Absolute ethyl alcohol：2.5h；+ 50% clarifier of 50% absolute ethyl alcohol：30min;Clarifier：2.5h；50% clarifier
+ 50% paraffin：30min；Paraffin：5h；(3) embed：Dewatered tissue is put into steel embedded box, is added and melted with embedding machine
Paraffin, and be frozen into wax stone on refrigeration platform, be put into 4 DEG C of refrigerators and preserve or cut into slices；(4) cut into slices：Wax stone is fixed
On paraffin slicing machine, it is whole by wax stone equating to be first adjusted to 20 μm of thickness, exposes tissue block, then with 4 μm of thickness section；
(5) patch：The section scaled off can be swum on 37 DEG C of the water surface, and section is reaped to the position of slide center with clean glass slide
Put, be subsequently put on 50 DEG C of baking piece machines and dry, and carry out mark.Section after drying is put into 4 DEG C of refrigerators and preserves or dyed；
(6) dewax：The glass slide for having paraffin section is put into 10min in clarifier, can be repeated once；(7) rehydration：Glass slide is existed
100%th, respectively immersion 2min, and shaking frequently in 95%, 85%, 70%, 50% ethanol, PBS and pure water；(8) HE is dyed：It will carry
Slide is put into 1-5min in haematoxylin, pure water rinsing 1min, adds activator 30s, pure water rinsing 1min, eosin stains 30s-
2min, tap water rinse 1min；(9) mounting：After slide natural air drying, 20 μ L resinenes of drop are used in glass slide sample center
Tweezers carefully cover glass slide, and have marked sample names information to observe.
3rd, immunohistochemical staining：(1)~(7) of step (1)~(7) with above step 2（Later step agents useful for same is equal
From immunohistochemical kit）；（8）2 drop Peroxidase Blocking are organizationally added dropwise in section preparation after air-drying
Reagent,5min；Slide is rinsed to no residual liquid, and 5min is rinsed with PBS；（9）Three drops are added dropwise on tissue block
Serum Blocking Reagent G （Close serum）15min is handled, serum remaining on slide is then got rid of and dries,
This step is rinsed without PBS；（10）3 drop Aridin Blocking Reagent are added dropwise on tissue block, handles 15min, is rushed with PBS
Wash 3 times, get rid of residual liquid and dry；（11）With Biotin Blocking Reagent（Biotin）15min is handled, then
Rinsed three times with PBS, get rid of residual liquid and dry；（12）The primary antibody covering for adding 80 μ L or so is incubated tissue block, in order to anti-
Only slide is dried, and can be put into the wet box of cotton ball of the inside equipped with soakage water, Cool Room 4 DEG C is overnight, this experiment uses
The primary antibody of MyHC；（13）After getting rid of residual liquid and drying, 3 drop Biotinylated Secondary are added dropwise to tissue block surface
Antibody（The secondary antibody of biotin labeling）, and 1h is placed at room temperature, and then rinsed 3 times with PBS, it is every all over 15min；（14）Get rid of
After falling residual liquid and drying, tissue block is incubated 30min plus 3 drop HSS-HRP, and is rinsed 3 times with PBS, every all over 2min；（15）
Get rid of after PBS residual liquids dry, developed the color 3-20min with AEC solution；（16）Use ddH2The slide that O immersions developed the color, processing
5min；（17）Slide tissue block 1~5min of haematoxylin dyeing after air-drying, pure water rinsing 1min, mounting after natural air drying, 4
DEG C preserve or micro- sem observation.
4th, Image Acquisition and data processing：（1）Image Acquisition：Fluorescence microscope is just put, using 10 × 40 times of times magnification
Number observation shooting, and mark scale；（2）Data processing：Each experiment is at least repeated 3 times, and as a result uses mean+SD
（Mean±SD）Represent, two groups of differences are calculated with Student ' s test, multigroup difference is examined with ANOVA.Mapping software is
GraphPad Prism 6, data analysis use SPSS（version 20）, statistical analysis uses two-sided test, differential expression side
Formula：*, p ＜ 0.05；*, p ＜ 0.01；* *, p ＜ 0.001.
The result is shown in Figure 1 of detection above, the results show that 18~28 days embryonic period, embryonic phases, three breeding pigs are produced without muscle fibre form
It is raw, arrive embryo 32 days, the primary muscle fibre of central circular occurs first in Wuzhi Mountain pig；During embryo 35 days, Lan Tang and length
There is primary muscle fibre in white pig, but the former quantity is significantly more than the latter, and the primary muscle fibre of Wuzhi Mountain pig continues to increase；
Embryo is arrived 42 days, three breeding pig primary muscle fibre numbers approach.
2 three breeding pig embryo of embodiment influences the transcript profile sequencing into oligogene/miRNA of flesh five periods
1st, sample collection：With 1 sample collection part of embodiment.
2nd, embryo RNA is extracted：（1）Embryo's meat sample tissue adds 1mL Trizol, is placed in fully shaking in historrhexis's instrument；
（2）Add 0.2 times of volume of chloroform in centrifuge tube and be vortexed and shake 15s, be stored at room temperature 3min；（3）Then by 4 DEG C of centrifuge tube,
12000g, centrifuges 15min；（4）It is divided into water phase after taking-up from top to bottom（Water white transparency）, intermediate layer（White）, organic phase（It is pink
Color）, the liquid of aqueous layer is transferred in new 1.5mL centrifuge tubes with pipettor；（5）Isometric shift to an earlier date is intervened into centrifuge tube
The isopropanol of precooling, overturns and mixes, be placed in -20 DEG C, 30~60min；（6）Then by centrifuge tube at 4 DEG C, 12000g, centrifugation
15min；（7）Centrifuge tube is taken out, white precipitate occurs in bottom at this time, and abandoning supernatant is clear using 75% ethanol of 1mL precoolings
Precipitation is washed, then at 4 DEG C, 7500g, centrifuges 5min；（8）Repeat step（7）Once；（9）Abandon supernatant, air drying precipitation 5
~10min；（10）The dissolving of 10 μ L RNase-free water is added, -80 DEG C of refrigerators preserve after surveying concentration.
3rd, transcript profile sequencing experimental procedure：After extracting sample total serum IgE and digesting DNA using DNase I, with Oligo
（dT）Enrichment with magnetic bead eukaryote mRNA；Addition interrupts reagent thermophilic in Thermomixer and mRNA is broken into short-movie section,
Using the mRNA after interrupting as one chain cDNA of templated synthesis, then prepare two chain synthesis reaction systems and synthesize two chain cDNA, and use
Kits recycling, cohesive end reparation, the 3 ' ends of cDNA add base " A " and jointing, and it is big then to carry out fragment
Small selection, finally carries out PCR amplification；The library built Agilent 2100 Bioanalyzer and ABI
After StepOnePlus Real-Time PCR System quality inspection qualifications, Illumina HiSeq are usedTM2000 or other sequencing
Instrument is sequenced.
4th, information analysis flow：By Illumina HiSeqTMThe data of 2000 sequencing gained are known as raw reads or raw
Data, then will carry out Quality Control (QC), to determine whether sequencing data is suitable for subsequent analysis to raw reads.After Quality Control, warp
Clean reads are obtained by filtration, are compared clean reads to reference sequences with SOAPaligner/SOAP2.After comparison,
With distribution situations and coverage of the reads on reference sequences, it is used as and judges comparison result whether by second of Quality Control
Index.If by second of Quality Control, the subsequent analysis such as gene expression are carried out.
5th, transcript profile data analysis：The calculating of gene expression amount usesRPKMMethod (Reads per kilobase
Transcriptome per million mapped reads), its calculation formula is：
The expression quantity that RPKM (X) is gene X is assumed in formula, then C is unique reads numbers compared to gene X, and N is unique
The reads sums of reference gene are compared, L is the base number of gene X code areas.RPKM methods are as a standardization, energy
The influence of sequencing amount difference and mrna length to calculating gene expression is eliminated, the gene expression amount being calculated can be used for directly
Connect the gene differential expression of the different sample rooms of comparison.
More than testing result see Fig. 2, the results showed that three interracial gene expressions occur significantly and meat yield
The gradient difference opposite sex rule being consistent.Promote into the gene of flesh or the initiate table of miRNA up to generally most earliest in the Wuzhi Mountain, and
Landrace expression time is later, such as MyoD, MyoG, miR-133b.And the gene suppressed into flesh then exists in embryonic development early stage
Expressed at most in Landrace, secondly blue pool pig, Wuzhi Mountain pig, such as ID2, MDFI and MSTN（28 dpc）.The mark of myotube differentiation
Will gene is also earliest, most in the expression of Wuzhi Mountain pig, and Landrace then occurs the latest, at least, such as MYH3, MYH13, DES, this
Result of one phenomenon also with Histomorphological before is coincide.
3 Wuzhi Mountain of embodiment and the quantitative qPCR and western blot of Landrace MyoD expressions are detected.
1st, sample collection：With example a sample collecting part.
2nd, embryo RNA is extracted：With 2 embryo's RNA extraction steps of example.
3rd, RNA reverse transcriptions obtain cDNA（Specific method refers to this area customary technical operation or various commercial kits
Specification）, cDNA is placed in -20 DEG C, and when use dilutes 5 times.
4th, real-time fluorescence quantitative PCR：Reaction system such as table 1：First add water in cDNA and mix, be then added in 384 orifice plates,
Then primer and 2 × SYBR Green qPCR Mix are mixed, added in corresponding hole, GAPDH is as reference gene, data
Analysis uses advanced relative quantification 2-∆∆Ct, quantify primer and be shown in Table 2.Added after 1 reaction system of table is mixed in 384 orifice plates, 3000
Leave heart 2min；Quantitative fluorescent PCR is carried out on LightCycler480 II, response procedures are：95 DEG C of pre-degeneration, 10min；
95 DEG C of denaturation, 5s；60 DEG C of annealing, 1min；72 DEG C of extension, 30s；40 circulations.Analyze solubility curve：95 DEG C, 5s；65 DEG C,
15s；95 DEG C, 0s.
Table 1 is the reaction system of qPCR
Table 2 is qPCR primer sequences
| Gene | Sense primer（5' to 3'） | Anti-sense primer（5' to 3'） |
| GAPDH | GCCTCCAAGGAGTAAGAAAC | GAAATTGTGAGGGAGATGCT |
| MyoD | ACCGCTCCGCGACGTAGATT | GCGAGTGTTCCTCGGGCTTT |
5th, Western blot are detected, and are comprised the following steps that：（1）The extraction of histone：A small amount of tissue block is placed in addition magnetic
In historrhexis's pipe of pearl, shredded before adding with scissors, add the protein lysate that 400 μ L contain PMSF and managed in historrhexis
In, it is homogenized, is subsequently placed on ice on historrhexis's instrument, is repeated several times making tissue pulverize as far as possible, cracks 30min；It will split
Solution liquid is moved in 1.5mL centrifuge tubes, and centrifuges 5min at 12,000rpm4 DEG C, then with 0.5mL centrifuge tubes packing supernatant
It is placed in -20 DEG C of preservations；（2）Protein quantification：Protein sample concentration is carried out using Coomassie Brilliant Blue homogeneous.5 are diluted first
× Coomassie brilliant blue to 1 ×, ELISA Plate adds the Coomassie brilliant blue liquid of 180 μ L per hole, and each sample is repeated 3 times.At room temperature
Shake 30s to mix, use the wavelength of 595nm to survey light absorption value after standing 5min, draw standard curve and calculate protein concentration；（3）Albumen
Sample electrophoresis：5 × albumen sample-loading buffer, 100 DEG C of heating 10min of dry type thermostat are added in the centrifuge tube for put protein sample
Denatured protein.Each hole adds 10 ~ 30 μ g protein samples（Determined according to protein concentration and expression）, deposition condition：
80V, 45min；120V, 80min；（4）Transferring film：Half-dried turn, the filter paper sheared is infiltrated in transferring film buffer solution first, then will
Pvdf membrane is put into methanol activation 1min, and positive plate is sequentially placed into filter paper, SDS-PAGE glue, pvdf membrane, filter paper from top to bottom（Every layer
There is not bubble）, cover minus plate, transferring film condition：20V, 60min；（5）Closing：Pvdf membrane is put into confining liquid after transferring film
（5% skim milk that TBST is prepared）When room temperature concussion closing 1 is small or Cool Room 4 DEG C is stayed overnight；（6）Primary antibody is incubated：Film is put into dilution
In good primary antibody, Cool Room 4 DEG C is overnight, is washed 3 times with TBST on shaking table afterwards, each 10min；（7）Secondary antibody is incubated：Film is put into
The secondary antibody diluted（1:5000）In, 1h is incubated at room temperature, is washed 3 times with TBST on shaking table again afterwards（Each 10min）；（8）With filter
Paper blots the water on film, is taken pictures after then covering ECL nitrite ion lucifuges 1min with ECL colour developing instrument.
Testing result above is shown in Fig. 3, the results show that 18 ~ 28 days embryonic period, embryonic phases, three breeding pigs are produced without muscle fibre form, arrives
Embryo 32 days, there is the primary muscle fibre of central circular first in Wuzhi Mountain pig；During embryo 35 days, the blue pool and Landrace are equal
There is primary muscle fibre, but the former quantity is significantly more than the latter, and the primary muscle fibre of Wuzhi Mountain pig continues to increase and diameter
Increase；Embryo is arrived 42 days, three breeding pig primary muscle fibres continue to break up.
MyoD Inhibit proliferatons are overexpressed in 4 C2C12 cells of embodiment
1st, transfect：（1）For the day before transfection by C2C12 cell inoculations in 6 orifice plates, density is about 30% ~ 50%, is incubated at complete culture
Base；（2）Transfection reagent is prepared：With miRmimic the or 10ul inhibitor of 250ul OPti-MEM dilutions 5ul and corresponding
Control miRNC；Another 250ul OPti-MEM dilution 5ul lipofectamine2000TM, place 5 minutes at room temperature；（3）
Will（2）In the two gently mix in incubation at room temperature 15 minutes, formed transfection composite；（4）Will（3）In transfection composite add
Into cell, add culture medium, gently mix, 6 it is small when after replace fresh culture.
2nd, real-time n cell detection：Utilize real-time n cell analyzer（Real-Time Cell
Analyzer）Analyze the propagation of cell.Instrument testing is calibrated：Preheater apparatus, then breeds detection plate（E-Plate 16）Per hole
In plus 50 μ L contain the DMEM of 10% FBS, reinstall instrument, adjust baseline.Cell Proliferation detects：By processed cell dissociation meter
Number is added in propagation detection plate, adds 8000 cells per hole, and the final DMEM with 10% FBS is by the culture medium polishing in every hole
180ul, 3 repetitions of every group of experiment.Set 5min to test once, cell Proliferation curve is drawn after length of testing speech 72h.
3rd, EDU propagation detection：（1）EDU presses 1 with 10% DMEM culture mediums:1000 dilution proportion, 12 orifice plates are per hole
300ul is added, 2h is incubated in cell incubator；（2）Washed 2 times, 5min/ times with the PBS of 500ul per hole；（3）Per hole 500ul
4% paraformaldehyde room temperature fixes 10min；（4）PBS is washed 2 times, 5min/ times；（5）TritonX- per hole 500ml 0.5%
100 incubation at room temperature 10min；（6）Repeat step（4）；（7）300ul/ holes Apollo staining reaction liquid（Prepared according to explanation）, room
Warm lucifuge is incubated 30min；（8）PBS washes 1 time, 5min；（9）At room temperature, the dyeing 5min per hole 300ul DAPI；（10）PBS cleaning
3 times, 5min/ times；（11）Take pictures under inverted fluorescence microscope.
4th, the PI cell cycles are detected：（1）The previous day, gets off cell dissociation loaded in 1.5ml EP pipes, with PBS cleaning,
Remove culture medium and pancreatin；（2）The ethanol of 70% precoolings of 1ml is added into every pipe, 4 DEG C of fixations are overnight；（3）1500r/s centrifugations 5
Minute collects cell and removes ethanol, is washed 3 times, every time 5 minutes with PBS；（4）1ml PI working solutions are added per hole, room temperature lucifuge is incubated
Educate 30min.（5）Machine testing in filtering.
5th, real-time fluorescence quantitative PCR:(1) cell total rna extracts：With reference to TRIzol extraction methods commonly used in the art or
Use commercialized extracts kit；（2）RNA is reversed to cDNA：Reference implementation example 3；（3）SYBR Green Ι Real-time
PCR：Reference implementation example 3.
5th, vector construction：（1）Extraction of plasmid DNA：With reference to Genstar plasmid extraction kit specifications.
（2）The acquisition of target gene and linearized vector：A:PCR：Reaction system is shown in Table 3, and template is C2C12 cell DNAs.
Response procedures are：95 DEG C of pre-degeneration, 30s；95 DEG C of denaturation, 10s；Annealing, 68 DEG C of extension, 2min（According to gene size and enzyme
Rate of amplification）；72 DEG C of extension, 5min；39 circulations.B:Synthesize fragment annealing:The single stranded DNA of chemical synthesis needs annealing to become
Double-strand, reaction system are shown in Table 4.Cycle of annealing is 95 DEG C, 10min；85 DEG C, 1min；75 DEG C, 1min；65 DEG C, 1min；55 DEG C,
1min；45 DEG C, 1min；30 DEG C, 1min.C:Carrier double digestion and connection：Using restricted restriction endonuclease of adjusting by carrier double digestion
Make its linearisation, endonuclease reaction system with reference to 5,37 DEG C of water-bath digestions 4 of table it is small when after Ago-Gel recycle, by the linear of acquisition
Change 16 DEG C of water-baths after carrier is prepared with genetic fragment by 6 system of table to connect overnight.D:Conversion and identification：(1) from -80 DEG C of refrigerators
DH5 α competent cells are taken out, connection product is added in super-clean bench after melting on ice, gently concussion makes its mixing, ice bath
30min；Ice bath 2min immediately after (2) 42 DEG C of heat shock 45s；(3) 600 μ L nonreactive LB fluid nutrient mediums of often pipe addition, 37 DEG C,
220rpm shake cultures 30min；Supernatant concentration thalline is abandoned in (4) 3000 × g, 1min centrifugation, and thalline has been spread evenly across accordingly
On the LB solid medium tablets of antibiotic, when 37 DEG C of inversion culture 12-16 are small；(5) picking monoclonal colony inoculation is in 600 μ
In LB fluid nutrient mediums of the L containing antibiotic, 37 DEG C, 220rpm shake cultures 5 it is small when, bacterium solution PCR and through positive gram of sequencing identification
It is grand.E:Western blot are tested：(1) sample treatment：With 2 times cells of PBS cleaning, 150~300 are added after blotting residual liquid
Protein lysates of the μ l containing PMSF, cell is stirred well on ice and is cracked completely, is transferred in 1.5ml centrifuge tubes and is used 1ml
Syringe is blown and beaten no longer sticky to sample repeatedly.(2) remaining Western blot operating procedure reference is shown in 3 phase of embodiment inside the Pass
Hold.F:Data processing and statistical analysis：Except special instruction, each biological experiment is at least repeated 3 times, experimental result articles for use average
± standard error（Mean±SEM）Represent；Experiment mapping uses 6 softwares of Prism, and data statistic analysis uses SPSS
（version 21）, the statistical analysis between two groups of data uses two-sided test, and the statistical analysis between three groups of data above uses
ANOVA, and be expressed as：Significant difference *：p<0.05；Pole significant difference * *：p<0.01；***：p<0.001.
Table 3 is PCR reaction systems
| Component | Volume（μL） |
| 5×PCR buffer（Mg2+） | 10 |
| dNTP | 4 |
| Template DNA | 1000ng |
| PrimeSTAR HS DNA polymerase | 0.5 |
| Forward Primer | 1 |
| Reverse Primer | 1 |
| H2O | Add to 50μL |
Table 4 is single-stranded annealing reaction system
| Component | Volume（μL） |
| Forward Fragment | 2 |
| Reverse Fragment | 2 |
| 10×T4 ligase Buffer | 2 |
| T4 PNK | 1 |
| H2O | Add to 20uL |
Table 5 is double digestion system
| Component | Volume（μL） |
| Plasmid DNA | 2ug |
| 10×fermentas Buffer | 5 |
| Enzyme 1 | 1 |
| Enzyme 2 | 1 |
| H2O | Add to 50 |
Table 6 is coupled reaction system
| Component | Volume（μL） |
| 10× T4 ligase buffer | 1 |
| Vector Fragment | 20ng |
| DNA Fragment | 80ng |
| T4 ligase | 0.3 |
| H2O | Add to 10 |
Above testing result is shown in Fig. 4, and the results show, which is overexpressed MyoD, can suppress the propagation of C2C12 cells.
The myotube that 5 C2C12 cellular proliferative stages of example example more early overexpression MyoD is produced after then breaking up is fewer
1st, induction differentiation：Treat that cell covers with completely, abandon culture medium, change the DMEM medium cultures containing 2% horse serum into, every two days
Replace a subculture.
2nd, immunofluorescence：（1）Cell fixes 10min with 4% pair of polyformaldehyde room temperature；（2）0.5% tritonX-100 room temperatures
It is incubated 10min；（3）30min is closed with 3%BSA；（4）It is incubated overnight with the diluted primary antibodies of 3%BSA at 4 DEG C；（5）Suck primary antibody,
3 5min are washed with PBS；（6）1 h is incubated with corresponding fluorescence secondary antibody room temperature lucifuge；（7）Secondary antibody is sucked, is washed with PBS
Wash 35 min；（8）DAPI dyes 5min；（9）PBS washes 35 min；（10）Take pictures under fluorescence microscope.
3rd, Western blot and real-time fluorescence quantitative PCR：Reference implementation example 4.Above testing result is shown in Fig. 5, as a result shows
Show that MyoD is overexpressed in the C2C12 cell Proliferation stages can suppress myogenic differentiation process, more early overexpression MyoD, the myotube of generation is got over
It is few.
MyoD is overexpressed in 6 NIH3T3 cells of embodiment can Inhibit proliferaton
1st, induction differentiation：5 relevant portion of reference implementation example.
2nd, immunofluorescence：5 relevant portion of reference implementation example.
3rd, Western blot and real-time fluorescence quantitative PCR：4 relevant portion of reference implementation example.
4th, real-time n cell detection, EDU dyeing and vector construction：4 relevant portion of reference implementation example.
Above testing result is shown in Fig. 6 and Fig. 7.
Claims (4)
1. postpone application of the expression time of Pig embryos phase myogenic differentiation factor M yoD in pig meat yield is improved.
2. a kind of improve the method for lean pork yield by postponing the expression time of gene, it is characterised in that postpones Pig embryos
The expression time of phase myogenic differentiation factor M yoD, so as to improve pig meat yield.
3. according to the method described in claim 2, it is characterized in that, postpone MyoD genes expresses 1 week to reach increase in pig
The effect of meat yield.
4. the according to the method described in claim 2, it is characterized in that, table for postponing Pig embryos phase myogenic differentiation factor M yoD
Method up to the time is as follows：The technology of MyoD genes is knocked in using the dox Cre-LoxP induced in local pig breed, is passed through
Cre-LoxP pigs and the MyoD of CRISPR-Cas9 technique constructions knock in the MyoD-LacZ-Stop-Cas9-KI that pig hybridizes
Pig；Or is induced by the sequential of MyoD gene expressions, makes local pig breed MyoD genes by sow feeding dox in different embryonic stages
Expression time postpones；Or the slow virus interference carrier of the RNA perturbation techniques structure MyoD genes using lentivirus mediated, in difference
Embryonic stage is to the sow injecting lentivirus interference carrier of local pig breed so as to postpone the expression time of MyoD.
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| PCT/CN2017/089556 WO2018196143A1 (en) | 2017-04-28 | 2017-06-22 | Method for increasing lean pork yield by delaying expression time of gene |
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| US5940198A (en) * | 1995-12-22 | 1999-08-17 | U.S. Philips Corporation | Optical unit for synchronizing clock signals |
| CA2946881A1 (en) * | 2014-04-28 | 2015-11-05 | Recombinetics, Inc. | Multiplex gene editing in swine |
| CN104388465A (en) * | 2014-09-24 | 2015-03-04 | 华南农业大学 | Application of MDFI in regulation of pig skeletal muscle growth and development |
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Non-Patent Citations (5)
| Title |
|---|
| LUIZ L. COUTINHO等: "Delayed somite formation in a quail line exhibiting myofiber hyperplasia is accompanied by delayed expression of myogenic regulatory factors and myosin heavy chain", 《DEVELOPMENT》 * |
| M.F.W. TE PAS等: "Influences of myogenin Genotypes on birth weight， growth rate， carcass weight， backfat thickness， and lean weight of pigs", 《JOURNAL OF ANIMAL SCIENCE》 * |
| MARCO CRESCENZI等: "MyoD induces growth arrest independent of differentiation in normal and transformed cells", 《PNAS》 * |
| QIN RF等: "Regulation of skeletal muscle differentiation in fibroblasts by exogenous MyoD gene in vitro and in vivo", 《MOL CELL BIOCHEM.》 * |
| SABOURIN LA等: "The molecular regulation of myogenesis", 《CLINICAL GENETICS》 * |
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