WO2018196143A1 - 一种通过延迟基因的表达时间来提高猪瘦肉产量的方法 - Google Patents

一种通过延迟基因的表达时间来提高猪瘦肉产量的方法 Download PDF

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WO2018196143A1
WO2018196143A1 PCT/CN2017/089556 CN2017089556W WO2018196143A1 WO 2018196143 A1 WO2018196143 A1 WO 2018196143A1 CN 2017089556 W CN2017089556 W CN 2017089556W WO 2018196143 A1 WO2018196143 A1 WO 2018196143A1
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myod
gene
pigs
pig
expression
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莫德林
张续勐
陈瑶生
刘小红
何祖勇
丛佩清
邱柏钦
郭慈琳
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Sun Yat Sen University
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Definitions

  • the present invention relates to the field of molecular genetics and, in particular, to a method for increasing lean meat production by delaying gene expression time.
  • primary muscle fibers Compared with pigs of different breeds, the number of primary muscle fibers and the ratio of secondary muscle fibers to primary muscle fibers of larger white pigs were significantly higher than those of small pigs. Therefore, between different body types of pigs, primary muscle fibers may have a greater impact on the total number of muscle fibers. In summary, primary muscle fibers play a decisive role in the amount of meat produced after birth in pigs with different meat production.
  • the family of myogenic regulatory factors is a family of genes that play a decisive role in myogenic processes.
  • the family has four members: MyoD, Myf5, Myogenin, and MRF4, which are characterized by a highly conserved basic helical loop helix domain with potential for myogenic regulation.
  • Myf5 is first expressed in the embryo and complements MyoD as the initial determinant of myoblasts.
  • Studies have found that transgenic mice lacking the above two genes do not form any muscle, but the loss of either of the above two genes alone will not have much effect on myogenic muscle, indicating that Myf5 and MyoD have certain functions. Overlap and Complementary.
  • Myoblasts isolated from knockout MyoD mice found that myoblasts knocked out of MyoD proliferated for a longer period of time, leading to a delay in differentiation. Recent studies have shown that the site of MyoD binding to Myf5 binding to the downstream gene is almost identical, but Myf5 lacks a stronger activation domain than MyoD, so there is no such strong ability to cause downstream gene expression as MyoD.
  • Zhao Xiao et al. used morphological observation and transcriptome sequencing of the longissimus dorsi muscle tissue from 35 days old embryos of Guangdong local pig breeds and foreign lean-type white pigs to 180 days after birth. It was found that the blue-tang pigs had primary muscle fibers in the embryos for 35 days, while the Changbai pigs had primary muscle fibers in the embryos for 49 days, but in the subsequent muscle development, the blue-tang pigs developed slower than the white pigs and were born. The amount of meat produced afterwards is also lower than the latter.
  • transcriptome sequencing they found that the myogenic determinant was expressed earlier in the early morning of the Lantang pig embryo than the Landrace pig, including the MyoD gene.
  • Yuqiang Zhao and others compared the other long-term muscle tissue of the local pig breed Tongcheng pig in western China and western lean pig breed Yorkshire pig (Large white pig) embryos for 30 days until 5 weeks after birth. Similar to the case of blue pond pigs, histomorphology found that Tongcheng pigs with less meat production also had higher numbers and higher density of myoblasts than Yorkshire pigs during the embryonic period of 30 days, but after the second In the development of round muscle, the secondary muscle fibers of England pigs develop faster than Tongcheng pigs.
  • the number of primary muscle fibers in the embryonic stage determines the total number of muscle fibers, and the total number of muscle fibers affects the amount of meat after birth. At the same time, the total number of muscle fibers has been determined during the embryonic stage. Therefore, the development of primary muscle fibers in the embryonic stage of pigs is decisive for the amount of meat after birth. influences.
  • the object of the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide a method for increasing the lean meat yield of pigs by delaying the expression time of the gene.
  • a method for increasing the lean meat yield of pigs by delaying the expression time of the gene, delaying the expression time of the myogenic differentiation factor MyoD in the pig embryo, thereby increasing the meat production of the pig.
  • the MyoD gene is postponed for one week in pigs to achieve an effect of increasing meat production.
  • the present invention simulates the early expression of MyoD gene in Chinese local pig breeds in a mouse-derived C2C12 myoblast cell line.
  • the expression of MyoD gene is 48 h and 24 h in advance, respectively, and the number of myotubes produced by differentiation is reduced, and the earlier expression is The fewer the number of myotubes produced.
  • we postpone the MyoD gene in pigs based on the actual results of a one-week expression of lean-type Landrace pigs compared to the small pig Wuzhishan pig MyoD. Expressed for 1 week to achieve an effect of increasing meat production.
  • the invention adopts the longest muscle-like tissue of the long-term muscles of the Changbai, Lantang and Wuzhishan pig embryos at the early 18th, 21st, 28th, 32nd, 35th and 42th day of the embryo production, and the myotube marker protein detection is performed.
  • the time of occurrence of primary muscle fibers of three breeds of pigs was found to be different: Wuzhishan pig first appeared primary muscle fibers in embryos 32 days, and blue pond and Landrace pigs showed primary muscle fibers in embryos 35 days, while the number of primary muscle fibers of Lantang pigs was significantly higher than that. Landrace pig.
  • MyoD was the earliest and highest in the Wuzhishan pig with the lowest meat production (the embryo began to express at 21 days), and in the Changbai pig with the most meat production. The expression was the latest and lowest (the embryo began to express in 28 days).
  • MyoD expression time we verified the expression of MyoD and the expression time in the C2C12 and NIH3T3 cell lines by overexpressing the MyoD plasmid, and the results were completely in line with expectations, ie in two Overexpression of MyoD in a cell line inhibits cell proliferation and reduces myotubes produced after differentiation.
  • a method for delaying the expression of MyoD is a technique for utilizing dox-induced Cre-LoxP knock-in of the MyoD gene in local pig breeds.
  • MyoD-LacZ-Stop-Cas9-KI pigs obtained by crossing MyoD knock-in pigs constructed by Cre-LoxP pigs with CRISPR-Cas9 technology, feeding dox to sows at different embryonic stages, induced the timing of MyoD gene expression, The local pig breed MyoD gene expression time was delayed.
  • a lentiviral-mediated RNA interference technique a lentiviral interference vector for constructing the MyoD gene, was used to inject a lentiviral interference vector into a sow of a local pig breed at different embryonic stages to delay the expression of MyoD.
  • the result of these methods is that the myoblasts have more time to proliferate, which in turn differentiates more muscle fibers and increases lean meat production in local pig breeds.
  • the present invention has the following beneficial effects:
  • the invention finds for the first time that the MyoD of pigs with higher meat production is later expressed, and the time of primary muscle fibers is also late, so that myoblasts have more time to proliferate, and the number of muscle fibers produced after differentiation is smaller than that of pigs with less meat production. There are many species, and eventually the amount of meat produced after birth is more. Based on this important finding, the present invention can affect or even change the expression time of MyoD in an animal by biotechnological means, thereby obtaining a new breed of livestock and poultry with a large amount of meat.
  • Figure 2 shows the transcriptome sequencing results of major genes/miRNAs affecting myoblasts in five stages of three breeds of pig embryos.
  • A-B MRFs and MEFs family genes;
  • C marker genes during myogenic differentiation;
  • D genes that inhibit myoblasts;
  • E myogenic-specific miRNAs that promote myoblasts.
  • Figure 4 is a graph showing the efficiency of overexpression of the MyoD gene.
  • D The proportion of proliferating cells in total cells.
  • E Overexpression of the MyoD gene affects the cell cycle.
  • Figure 5 is the effect of overexpression of the MyoD gene on myogenic differentiation at different time points.
  • A The MyoD gene was overexpressed 2 days before differentiation (-2d) and 1 day before differentiation (-1d), and the pCMV empty plasmid was used as a negative control. At the same time, differentiation was induced, and the expression level of MYHC protein was detected by Western-blot.
  • B Overexpression of MyoD gene at different time points, myotube immunofluorescence results.
  • C Quantitative results of gray scale scanning of MYHC protein for Western-blot of Figure A.
  • Figure 6 is a diagram of overexpression of MyoD to fuse NIH3T3 cells to form myotubes.
  • A After overexpressing MyoD, the mRNA expression level of MyoD was examined.
  • B After 48 h of transfection, the level of MyoD protein was measured.
  • C Detection of mRNA levels of key factors of myogenic differentiation after 7 days of induced differentiation.
  • D After induction of differentiation for 7 days, myotube formation was observed under white light.
  • E After overexpression of MyoD, differentiation was induced for 7 days, and the expression of MyHC was detected by immunofluorescence.
  • F After overexpression of MyoD, differentiation was induced for 7 days, and the expression of MyHC was detected at the protein level. .
  • *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, n 3.
  • Ruler size 100 ⁇ m.
  • Figure 7 is a graph overexpressing MyoD and inhibiting proliferation of NIH3T3 cells.
  • A Real-time label-free cell detection system detects the proliferation of NIH3T3 cells compared with the control group after overexpression of MyoD.
  • B Count the number of cells in the control group and the overexpression group at different times of GM.
  • C After over-expression of MyoD, PI staining Color, flow cytometry analysis of cell cycle, overexpression of MyoD compared with the control group, NIH3T3 cells G1 arrest, S phase cells decreased.
  • D Immunofluorescence detection of EdU positive cells and statistics.
  • test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used are, if not specified, commercially available reagents and materials.
  • the three breeds of pigs are abbreviated as: Changbai pig (LR), Lantang pig (LT) and Wuzhishan pig (WZS); the six embryonic periods are respectively abbreviated as: 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) and 42 days (LR6/LT6/WZS6).
  • paraffin section and HE staining (1) fixed: 4% paraformaldehyde placed for 24 to 48 hours; (2) dehydration: the sample is placed in the embedding box, marked, and then placed in a fully automatic dehydrator , the dehydration step is: 70% ethanol: 2h; 80% ethanol: 2h; 95% ethanol: 30min; anhydrous ethanol: 2.5h; 50% absolute ethanol + 50% transparent agent: 30min; transparent agent: 2.5h; % clearing agent + 50% paraffin wax: 30min; paraffin wax: 5h; (3) embedding: the dehydrated tissue is placed in a steel embedding box, the melted paraffin is added by the embedding machine, and solidified into wax on the refrigeration platform.
  • the dried slices are stored in a refrigerator at 4 ° C or dyed; (6) Dewaxing: slides with paraffin sections are placed in a clearing agent for 10 min, which can be repeated once; (7) Rehydration: Slides Soaked in 100%, 95%, 85%, 70%, 50% ethanol, PBS and pure water for 2min, occasionally oscillate; (8) HE staining: put the slide into hematoxylin 1-5min, pure Wash water for 1 min, add activator for 30s, rinse with pure water for 1min, eosin stain for 30s-2min, rinse with tap water for 1min; (9) Cover: After the slide is naturally air-dried, drop 20 ⁇ L of neutral resin in the slide specimen center, use The tweezers carefully cover the slide and label the sample name for observation.
  • Steps (1) to (7) are the same as steps (1) to (7) above (the reagents used in the subsequent steps are all from the immunohistochemistry kit); (8) the sliced specimen is air-dried in the tissue. Two drops of Peroxidase Blocking Reagent were added dropwise for 5 min; the slides were rinsed to the absence of residual liquid and rinsed with PBS for 5 min; (9) Three drops of Serum Blocking Reagent G (blocked serum) were added to the tissue block for 15 min, then the glass was removed.
  • image acquisition and data processing (1) image acquisition: upright fluorescence microscope, using 10 ⁇ 40 times magnification to observe the shooting, and label the ruler; (2) data processing: each experiment at least 3 times, the results used Mean ⁇ standard deviation (Mean ⁇ SD) indicates that the difference between the two groups was calculated using Student's test, and multiple sets of differences were examined by ANOVA.
  • the mapping software was GraphPad Prism 6, the data analysis was SPSS (version 20), the statistical analysis was performed by two-sided test, and the differential expression was: *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001.
  • Example 2 Transcriptome sequencing of major genes/miRNAs affecting myoblasts in five stages of three breeds of pig embryos
  • Sample collection same as the sample collection part of Example 1.
  • embryonic RNA extraction (1) embryonic meat-like tissue added 1mL Trizol, placed in the tissue disruptor to fully oscillate; (2) add 0.2 times the volume of chloroform in the centrifuge tube and vortex for 15s, rest at room temperature for 3min; 3) Then centrifuge the tube at 4 ° C, 12000g, centrifuge for 15min; (4) take out from top to bottom into the water phase (colorless transparent), the middle layer (white), the organic phase (pink), with a pipette Transfer the liquid in the aqueous layer to a new 1.5 mL centrifuge tube; (5) Inject an equal volume of pre-cooled isopropanol into the centrifuge tube, mix by inversion, and place at -20 ° C for 30-60 min; (6) Then centrifuge the tube at 4 ° C, 12000g, centrifuge for 15min; (7) take out the centrifuge tube, there will be a white precipitate at the bottom, discard the supernatant, wash the pellet with 1mL of
  • transcriptome sequencing experimental steps extract the sample total RNA and digest the DNA with DNase I, then enrich the eukaryotic mRNA with magnetic beads with Oligo (dT); add the interrupting reagent to interrupt the mRNA at the appropriate temperature in the Thermomixer A short fragment was obtained, and the stranded cDNA was synthesized by using the broken mRNA as a template, and then a two-stranded synthesis reaction system was used to synthesize the double-stranded cDNA, and the kit was used for purification and recovery, sticky end repair, and the 3' end of the cDNA plus the base. A "connection joint and then fragment size selection, the final PCR amplification; library were used and after Agilent 2100Bioanalyzer ABI StepOnePlus Real-Time PCR System quality inspection, using Illumina HiSeq TM 2000 or other sequencer for sequencing.
  • the information analysis process data 2000 obtained by the sequencing Illumina HiSeq TM or called raw reads raw data, for raw reads subsequently proceeds quality (the QC), to determine whether the data is suitable for subsequent sequencing analysis.
  • quality control clean reads are obtained by filtering, and clean reads are compared to the reference sequence using SOAPaligner/SOAP2 [16] .
  • the distribution and coverage of the reads on the reference sequence are used as indicators for judging whether the comparison result passes the second quality control. If the second quality control is passed, subsequent analysis such as gene expression is performed.
  • transcriptome data analysis the calculation of gene expression using RPKM [17] method (Reads per kilobase transcriptome per million mapped reads), the calculation formula is: In the formula, RPKM(X) is assumed to be the expression level of gene X, then C is the only number of reads aligned to gene X, N is the only total number of reads aligned to the reference gene, and L is the number of bases of the gene X coding region. As a standardized treatment, RPKM method can eliminate the influence of sequencing difference and gene length on the calculation of gene expression. The calculated gene expression can be used to directly compare gene differential expression between different samples.
  • Sample collection same as the sample one sample collection part.
  • Embryo RNA extraction same as Example 2 embryo RNA extraction step.
  • RNA Reverse transcription of RNA to obtain cDNA (for specific methods, refer to the procedures of the prior art or various commercial kits), the cDNA is placed at -20 ° C, and diluted 5 times when used.
  • the reaction procedure was: pre-denaturation at 95 ° C for 10 min; denaturation at 95 ° C for 5 s; annealing at 60 ° C for 1 min; Extend 72 ° C, 30 s; 40 cycles.
  • the dissolution curve was analyzed: 95 ° C, 5 s; 65 ° C, 15 s; 95 ° C, 0 s.
  • Table 1 shows the reaction system of qPCR
  • Table 2 shows the qPCR primer sequence
  • Upstream primer (5'to 3') Downstream primer (5'to 3') GAPDH GCCTCCAAGGAGTAAGAAAC GAAATTGTGAGGGAGATGCT MyoD ACCGCTCCGCGACGTAGATT GCGAGTGTTCCTCGGGCTTT
  • tissue protein extraction a small number of tissue blocks placed in the tissue disruption tube added to the magnetic beads, cut with scissors before the addition, add 400 ⁇ L of PMSF containing protein lysate to the tissue In the crushing tube, homogenate on the tissue disrupter, then placed on ice, repeat the tissue as much as possible, and lyse for 30 min; transfer the lysate to a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 4 min at 5 °C. Then, the supernatant was dispensed with a 0.5 mL centrifuge tube and stored at -20 ° C; (2) Protein quantification: The protein sample concentration was uniform using the Coomassie Brilliant Blue method.
  • Transfection (1) C2C12 cells were inoculated into 6-well plates one day prior to transfection, with a density of approximately 30% to 50%, cultured in complete medium; (2) Transfection reagent preparation: diluted with 250 ul OPti-MEM 5ul of miRmimic or 10ul inhibitor and the corresponding control miRNC; another 250ul OPti-MEM diluted 5ul lipofectamine2000 TM and allowed to stand at room temperature for 5 minutes; (3) gently mix the two in (2) for 15 minutes at room temperature To form a transfection complex; (4) Add the transfection complex in (3) to the cells, add the medium, mix gently, and replace the fresh medium after 6 hours.
  • Real-time label-free cell detection Cell proliferation was analyzed using a Real-Time Cell Analyzer. Instrument calibration: Preheat the instrument, then add 50 ⁇ L of DMEM containing 10% FBS to each well of the proliferation assay plate (E-Plate16), replace the instrument, and adjust the baseline. Cell proliferation assay: The treated cell digestion count was added to the proliferation assay plate, and 8000 cells were added to each well. Finally, the culture medium of each well was filled with 180 ul in 10% FBS DMEM, and each group was repeated 3 times. The test was set for 5 min, and the cell proliferation curve was drawn after 72 h.
  • Instrument calibration Preheat the instrument, then add 50 ⁇ L of DMEM containing 10% FBS to each well of the proliferation assay plate (E-Plate16), replace the instrument, and adjust the baseline.
  • Cell proliferation assay The treated cell digestion count was added to the proliferation assay plate, and 8000 cells were added to each well. Finally, the culture medium of each well was filled with 180 ul in 10% FBS DMEM, and each
  • EDU proliferation assay (1) EDU is diluted with 1:1000 in 10% DMEM medium, 300 ul per well in a 12-well plate, and incubated for 2 h in a cell culture incubator; (2) Washed with 500 ul of PBS per well 2 times, 5min/time; (3) Each well was fixed with 500ul 4% paraformaldehyde for 10min at room temperature; (4) Washed twice with PBS for 5min/time; (5) 500ml with 0.5% tritonX-100 per well at room temperature Incubate for 10 min; (6) repeat step (4); (7) 300 ul / well Apollo staining reaction solution (according to instructions), incubate at room temperature for 30 min in the dark; (8) wash once with PBS for 5 min; (9) at room temperature, 300 ⁇ L DAPI staining per well for 5 min; (10) PBS washing 3 times, 5 min/time; (11) photographed under an inverted fluorescence microscope.
  • PI cell cycle detection (1) the day before, the cells were digested and placed in 1.5ml EP tube, washed with PBS, remove the medium and trypsin; (2) add 1ml 70% pre-cooled to each tube Ethanol was fixed overnight at 4 ° C; (3) Centrifuge for 5 minutes at 1500 r/s to collect ethanol, and wash with ethanol for 3 times for 5 minutes each time; (4) Add 1 ml of PI working solution per well, incubate at room temperature for 30 min in the dark. (5) Filter on machine detection.
  • RNA extraction from cells refer to the TRIzol extraction method commonly used in the art or use a commercially available extraction kit; (2) RNA inversion to cDNA: Reference Example 3; (3) SYBR Green ⁇ Real-time PCR: Refer to Example 3.
  • Plasmid DNA extraction Refer to the Genstar plasmid extraction kit instructions.
  • the annealing procedure was 95 ° C, 10 min; 85 ° C, 1 min; 75 ° C, 1 min; 65 ° C, 1 min; 55 ° C, 1 min; 45 ° C, 1 min; 30 ° C, 1 min.
  • C Double digestion and ligation of the vector: The vector was double-digested and linearized by restriction enzyme endonuclease. The digestion reaction system was digested with agarose gel at 37 ° C for 4 hours, and then recovered. The linearized vector and gene fragment were prepared according to the system of Table 6 and connected overnight at 16 ° C in a water bath.
  • Transformation and identification (1) DH5 ⁇ competent cells were taken out from the -80 °C refrigerator, melted on ice, and the ligation product was added to the clean bench, gently shaken to mix, ice bath for 30 min; (2) 42 ° C Immediately after heat shock for 45 s, ice bath for 2 min; (3) 600 ⁇ L of anti-LB liquid medium was added to each tube, and cultured at 37 ° C and 220 rpm for 30 min; (4) 3000 ⁇ g, 1 min, centrifuged to discard the supernatant, and the cells were removed.
  • Example 3 Refer to the relevant content of Example 3 for the rest of the Western blot operation steps.
  • F Data processing and statistical analysis: Unless otherwise specified, each biological experiment was repeated at least 3 times, and the experimental results were expressed as mean ⁇ standard error (Mean ⁇ SEM); experimental mapping using Prism 6 software, statistical analysis using SPSS ( Version 21), Statistical analysis between the two groups of data was performed using a two-sided test. Statistical analysis between the three groups of data was performed using ANOVA and expressed as: significant difference *: p ⁇ 0.05; extremely significant difference **: p ⁇ 0.01; :p ⁇ 0.001.
  • Table 3 shows the PCR reaction system
  • Table 4 is a single-strand annealing reaction system
  • Table 5 is a double enzyme digestion system
  • Induction of differentiation After the cells are completely overgrown, the medium is discarded and replaced with DMEM medium containing 2% horse serum, and the medium is changed every two days.

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Abstract

提供一种通过延迟基因的表达时间来提高猪瘦肉产量的方法，具体为通过推迟猪胚胎早期成肌决定基因MyoD表达时间，使得胚胎早期初级肌纤维形成时间推后，增加肌纤维总数，从而提高出生后瘦肉产量。

Description

一种通过延迟基因的表达时间来提高猪瘦肉产量的方法 技术领域
本发明涉及分子遗传技术领域，具体地，涉及一种通过延迟基因的表达时间来提高猪瘦肉产量的方法。
背景技术
猪的骨骼肌发育在胚胎期主要有两轮：1)初级肌纤维的形成阶段，妊娠后第14～22天是体节的发育，随后轴旁中胚层发育形成生肌节，初级肌纤维在妊娠后第35天左右开始出现，在妊娠第49天左右达到增殖高峰，并一直持续到妊娠第60天。2)次级肌纤维形成阶段，妊娠第45～90天左右次级肌纤维围绕初级肌纤维周围分化发育，并在妊娠75天左右达到高峰，妊娠第91天之后，肌纤维数目基本不再改变，肌纤维在出生后主要进行肌纤维的肥大以及类型变化的成熟过程。因此，猪胚胎期骨骼肌的肌纤维总数在出生前就已经确定。
前人研究发现，肌纤维数目和大小是产肉量的主要决定因素，这其中，肌纤维数目更加重要，因为大小适中的肌纤维比例较高的动物会产生更多的瘦肉和较好的的肉，并且肌纤维数目越多，动物个体生长的速度就越快越高效。比较大白和长白猪杂交后子一代不同大小的胚胎，发现初级肌纤维出现的时间没有差异，但是体型小的猪胚胎次级肌纤维的数量要显著少于体型大的胚胎，并且在出生后有17％的肌纤维总数的差异。因此在同一品种内，次级肌纤维可能对总肌纤维数目的影响更大。不同品种猪之间的比较，体型较大的大白猪初级肌纤维的数目以及次级肌纤维与初级肌纤维的比例都显著高于小型猪。因此，在不同体型猪品种间，初级肌纤维可能对总肌纤维数目的影响更大。综上所述，初级肌纤维在产肉量不同的猪种中，对出生后的产肉量起到决定性的作用。
成肌调控因子家族是成肌过程中起决定性作用的基因家族。该家族有四个成员：MyoD、Myf5、Myogenin和MRF4，其共同特征是拥有一个具有成肌调控潜力的高度保守的碱性螺旋环螺旋结构作用域。四个成员当中，Myf5在胚胎中最先表达，并与MyoD相互补充来作为成肌的起始决定基因。有研究发现缺失上述两个基因的转基因小鼠不会形成任何肌肉，但单独缺失上述两者中的任意一个基因，对成肌均不会有太大的影响，说明Myf5和MyoD有一定的功能重叠和 互补。在对敲除MyoD小鼠中分离的成肌细胞进行研究发现，敲除MyoD的成肌细胞会增殖更长的时间，导致分化的延迟。最近的研究结果显示，MyoD与Myf5结合下游基因的位点几乎一致，但与MyoD相比，Myf5缺少一个较强的激活作用域，因此没有MyoD那么强的引起下游基因表达的能力。
赵晓等在2011年，利用广东地方猪种蓝塘猪和国外瘦肉型长白猪胚胎35天到出生后180天的背最长肌组织进行了形态观察和转录组测序。结果发现，蓝塘猪在胚胎35天就已经出现了初级肌纤维，而长白猪到了胚胎49天才出现初级肌纤维，但是在之后的肌肉发育过程中，蓝塘猪肌肉发育要慢于长白猪，并且出生后产肉量也低于后者。通过转录组测序，他们发现成肌决定因子在蓝塘猪胚胎早期要比长白猪更早更多的表达，这其中就包括MyoD基因。
2015年，Yuqiang Zhao等人比较研究了另外一种华中地区的地方猪种通城猪与西方瘦肉猪种约克夏猪(大白猪)胚胎30天直到出生后5周的背最长肌组织。与蓝塘猪情况类似，组织形态学发现，产肉量较少的通城猪同样在胚胎期30天比约克夏猪拥有较多数量和较高密度的成肌细胞，但是在之后的第二轮肌肉发育中，约克夏猪的次级肌纤维的发育速度要快于通城猪。
猪胚胎期初级肌纤维的数量决定了肌纤维总数，而肌纤维总数又会影响出生后的产肉量，同时肌纤维总数在胚胎期已经确定，因此猪胚胎期初级肌纤维的发育对出生后产肉量具有决定性影响。
发明内容
本发明的目的是为了克服现有技术的上述不足，提供一种通过延迟基因的表达时间来提高猪瘦肉产量的方法。
为了实现上述目的，本发明是通过以下技术方案予以实现的：
延迟猪胚胎期成肌分化因子MyoD的表达时间在提高猪产肉量中的应用。
一种通过延迟基因的表达时间来提高猪瘦肉产量的方法，延迟猪胚胎期成肌分化因子MyoD的表达时间，从而提高猪产肉量。优选地，推迟MyoD基因在猪中表达1周以达到增加产肉量的效果。
本发明在小鼠来源的C2C12成肌细胞系中模拟了中国地方猪种中MyoD基因早表达的情况，分别提前MyoD基因表达48h和24h，均使分化产生的肌管数量减少，并且越早表达产生的肌管数量越少。在实际应用中，我们根据瘦肉型长白猪比小型猪五指山猪MyoD晚表达一周的实际结果，推迟MyoD基因在猪中 表达1周以达到增加产肉量的效果。
本发明使用产肉量依次递减的长白、蓝塘和五指山猪胚胎早期18、21、28、32、35和42天的背最长肌肉样组织进行组织形态学观察及肌管标志蛋白检测，我们发现三个品种猪初级肌纤维出现的时间存在差异：五指山猪在胚胎32天首先出现初级肌纤维，蓝塘和长白猪在胚胎35天出现初级肌纤维，而此时蓝塘猪的初级肌纤维数量显著高于长白猪。通过对上述样品进行转录组测序、定量PCR和Western blot分析，我们发现MyoD在产肉量最少的五指山猪中表达最早、最高(胚胎21天开始表达)，而在产肉量最多的长白猪中表达最晚、最低(胚胎28天开始表达)。为了验证myoD表达时间对成肌的影响，我们在C2C12和NIH3T3细胞系中通过过表达MyoD质粒来验证MyoD的表达以及表达时间是否会影响最终分化的肌纤维数量，而结果完全符合预期，即在两种细胞系中过表达MyoD均会抑制细胞的增殖，分化后产生的肌管减少。基于这一重要发现，我们可以通过生物技术手段来影响甚至是改变动物体内MyoD的表达时间，从而获得产肉量多的畜禽新品种。实现推迟表达MyoD的方法有在地方猪种中利用dox诱导的Cre-LoxP敲入MyoD基因的技术。通过Cre-LoxP猪与CRISPR-Cas9技术构建的MyoD敲入猪杂交得到的MyoD-LacZ-Stop-Cas9-KI猪，在不同的胚胎时期对母猪饲喂dox，诱导MyoD基因表达的时序，使地方猪种MyoD基因表达时间推迟。此外还有利用慢病毒介导的RNA干扰技术，即构建MyoD基因的慢病毒干扰载体，在不同胚胎时期对地方猪种的母猪注射慢病毒干扰载体从而推迟MyoD的表达时间。这些方法的结果就是使成肌细胞有更多的时间增殖，进而分化出更多肌纤维，提高地方猪种的瘦肉产量。
与现有技术相比，本发明具有如下有益效果：
本发明首次发现：产肉量较多的猪MyoD表达较晚，而且出现初级肌纤维时间也较晚，因此成肌细胞有更多的时间增殖，分化后产生的肌纤维数量比产肉量少的猪种多，最终在出生后产肉量也就更多。基于这一重要发现，本发明可以通过生物技术手段来影响甚至是改变动物体内MyoD的表达时间，从而获得产肉量多的畜禽新品种。
附图说明
图1是三品种猪胚胎早期初级肌纤维出现时间存在差异的实验验证图；A.HE染色，400×；B.免疫组化，400×；C.初级肌纤维的数量统计；D.三品种猪胚胎 背最长肌组织免疫蛋白印迹结果；免疫组化，WB实验抗体为MYHC，内参为GAPDH,箭头所指为初级肌纤维；标尺＝50μm；*，p＜0.05；**，p＜0.01。
图2是三品种猪胚胎五个时期影响成肌的主要基因/miRNA的转录组测序结果。(A-B)MRFs与MEFs家族基因；(C)成肌分化时期的标志基因；(D)抑制成肌的基因；(E)促进成肌的成肌特异miRNA。
图3为三品种猪胚胎早期初级肌纤维出现时间存在差异的实验验证图；A.HE染色，400×；B.免疫组化，400×；C.初级肌纤维的数量统计；D.三品种猪胚胎背最长肌组织免疫蛋白印迹结果。免疫组化，WB实验抗体为MYHC，内参为GAPDH,箭头所指为初级肌纤维；标尺＝50μm；*，p＜0.05；**，p＜0.01。
图4是过表达MyoD基因效率验证图。A.Western-blot检测MyoD蛋白过表达效果。B.Q-PCR定量检测MyoD基因过表达效果。结果显示构建的MyoD过表达载体无论是在蛋白水平还是mRNA水平都是十分有效的。C.过表达MyoD基因，利用EDU染色检测C2C12细胞增殖。D.正在增殖细胞占总细胞的比例。E.过表达MyoD基因对细胞周期的影响。F.过表达MyoD基因后，利用实时无标记细胞检测系统检测C2C12细胞增殖。G.过表达MyoD基因后，Q-PCR定量检测细胞周期相关因子基因的表达。
图5是不同时间点过表达MyoD基因对成肌分化的影响。A.分化前2天(-2d)和分化前1天(-1d)过表达MyoD基因，pCMV空载质粒为阴性对照。同时诱导分化，Western-blot检测MYHC蛋白的表达水平。B.不同时间点过表达MyoD基因，肌管免疫荧光结果图。C.为图A的Western-blot检测MYHC蛋白的灰度扫描定量结果。
图6是过表达MyoD使NIH3T3细胞融合形成肌管的图。(A)过表达MyoD后，检测MyoD的mRNA表达水平。(B)转染48h后，检测MyoD蛋白水平。(C)诱导分化7d后，成肌分化关键因子的mRNA水平检测。(D)诱导分化7d后，白光下观察到肌管形成。(E)过表达MyoD后，诱导分化7d，免疫荧光检测到MyHC的表达。(F)过表达MyoD后，诱导分化7d，蛋白水平检测到MyHC的表达。。*p<0.05，**p<0.01，***p<0.001，n＝3。标尺大小：100μm。
图7是过表达MyoD，抑制NIH3T3细胞的增殖的图。(A)实时无标记细胞检测系统检测过表达MyoD后，与对照组相比NIH3T3细胞的增殖情况。(B)统计GM不同时期对照组和过表达组的细胞数目。(C)过表达MyoD后，PI染 色，流式细胞术分析细胞周期，过表达MyoD后与对照组相比，NIH3T3细胞发生G1期阻滞，S期细胞减少。(D)免疫荧光检测EdU阳性细胞并统计。(E)细胞周期关键因子的mRNA水平检测。*p<0.05，**p<0.01，***p<0.001，n＝3。标尺大小：100μm。
具体实施方式
下面结合说明书附图和具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例1长白、蓝塘和五指山猪初级肌纤维出现时间的确定
1、样品采集：长白、蓝塘和五指山猪均根据各自系谱，挑选全同胞或半同胞的12头母猪，各使用同一头相应品种公猪的精液进行人工授精(五指山猪采用自然授精方式)，在相同的饲养条件下喂食相同的饲料。所有动物的处理办法都严格按照广东省动物保护与使用委员会批准的流程进行，批准文号为：SCXK(Guangdong)2011–0029和SYXK(Guangdong)2011–0112。在胚胎期18、21、28、32、35和42天分别屠宰各品种2头母猪，剖腹剪开子宫取出胚胎。18和21天由于胚胎较小故取整个胚胎，其余时期分离胚胎背部肉样组织，剪成约黄豆大小的组织块。所有的样品均用PBS洗干净，一部分装入2.0mL冻存管中，并迅速放入液氮保存；另一部分放入4％多聚甲醛溶液备用。为了便于标记，三品种猪分别简写为：长白猪(LR)、蓝塘猪(LT)和五指山猪(WZS)；6个胚胎时期分别简写为：18天(LR1/LT1/WZS1)、21天(LR2/LT2/WZS2)、28天(LR3/LT3/WZS3)、32天(LR4/LT4/WZS4)、35天(LR5/LT5/WZS)和42天(LR6/LT6/WZS6)。
2、石蜡切片和HE染色：(1)固定：4％多聚甲醛放置24～48小时；(2)脱水：将样品放入包埋盒中，做好标记，然后放入全自动脱水机中，脱水步骤为：70％乙醇：2h；80％乙醇：2h；95％乙醇：30min；无水乙醇：2.5h；50％无水乙醇+50％透明剂：30min；透明剂：2.5h；50％透明剂+50％石蜡：30min；石蜡：5h；(3)包埋：将脱水后的组织放入钢包埋盒中，用包埋机加入融化的石蜡，并在制冷平台上凝固成蜡块，放入4℃冰箱保存或进行切片；(4)切片：将蜡块固定在石蜡切片机上，先调整为20μm的厚度将蜡块修平整，露出组织 块，随后用4μm的厚度切片；(5)贴片：切下来的切片会漂浮在37℃的水面上，用洁净的载玻片将切片捞到玻片正中的位置，随后放在50℃烘片机上烘干，并做好标记。烘干后的切片放入4℃冰箱保存或进行染色；(6)脱蜡：将有石蜡切片的载玻片放入透明剂中10min，可重复一次；(7)复水：将载玻片在100％、95％、85％、70％、50％的乙醇，PBS以及纯水中各浸泡2min，并不时震荡；(8)HE染色：将载玻片放入苏木素中1-5min，纯水冲洗1min,加入活化剂30s，纯水冲洗1min，伊红染色30s-2min，自来水冲洗1min；(9)封片：玻片自然风干后，滴20μL中性树脂于载玻片标本中心，用镊子小心盖上载玻片，并标注好样本名称信息以便观察。
3、免疫组化染色：步骤(1)～(7)同以上步骤2的(1)～(7)(后面步骤所用试剂均来自免疫组化试剂盒)；(8)切片标本风干后在组织上滴加2滴Peroxidase Blocking Reagent,5min；冲洗玻片至没有残余液体，并用PBS冲洗5min；(9)在组织块上滴加三滴Serum Blocking Reagent G(封闭血清)处理15min，然后甩掉玻片上残余的血清并擦干，此步无需PBS冲洗；(10)组织块上滴加3滴Aridin Blocking Reagent，处理15min，用PBS冲洗3次，甩掉残余液体并擦干；(11)用Biotin Blocking Reagent(生物素)处理15min，随后用PBS冲洗三遍，甩掉残余液体并擦干；(12)加入80μL左右的一抗覆盖孵育组织块，为了防止玻片干燥，可放入里面装有浸润水的棉花球的湿盒中，4℃冷库过夜，本实验使用的是MyHC的一抗；(13)甩掉残余液体并擦干后，向组织块表面滴加3滴Biotinylated Secondary Antibody(生物素标记的二抗)，并在室温下放置1h，随后用PBS冲洗3遍，每遍15min；(14)甩掉残余液体并擦干后，组织块加上3滴HSS-HRP孵育30min，并用PBS冲洗3遍，每遍2min；(15)甩掉PBS残余液体擦干后，用AEC溶液显色3-20min；(16)用ddH2O浸泡显色过的玻片，处理5min；(17)风干后的玻片组织块用苏木素染色1～5min，纯水冲洗1min，自然风干后封片，4℃保存或显微镜观察。
4、图像采集及数据处理：(1)图像采集：正置荧光显微镜，采用10×40倍的放大倍数观察拍摄，并标注标尺；(2)数据处理：每个实验至少重复3次，结果用平均值±标准差(Mean±SD)表示，用Student’s test计算两组差异，用ANOVA检验多组差异。作图软件为GraphPad Prism 6，数据分析使用SPSS(version 20)，统计分析采用双侧检验，差异表达方式：*，p＜0.05；**，p＜0.01； ***，p＜0.001。
以上检测的结果见图1，结果显示，胚胎期18～28天三品种猪均没有肌纤维形态产生，到了胚胎32天，五指山猪首先出现了中心圆环状的初级肌纤维；胚胎35天时，蓝塘和长白猪均出现了初级肌纤维，但前者数量明显多于后者，而五指山猪的初级肌纤维继续增多；到了胚胎42天，三品种猪初级肌纤维数目接近。
实施例2三品种猪胚胎五个时期影响成肌的主要基因/miRNA的转录组测序
1、样品采集：同实施例1样品采集部分。
2、胚胎RNA提取：(1)胚胎肉样组织加入1mL Trizol，置于组织破碎仪中充分震荡；(2)加0.2倍体积氯仿于离心管中并涡旋震荡15s，室温静置3min；(3)随后将离心管4℃，12000g，离心15min；(4)取出后自上而下分为水相(无色透明)，中间层(白色)，有机相(粉红色)，用移液器将水相层的液体转移到新的1.5mL离心管中；(5)向离心管中介入等体积提前预冷的异丙醇，颠倒混匀，置于-20℃、30～60min；(6)随后将离心管在4℃，12000g，离心15min；(7)取出离心管，此时底部会出现白色沉淀，弃去上清液，使用1mL预冷的75％乙醇清洗沉淀，随后在4℃，7500g，离心5min；(8)重复步骤(7)一次；(9)弃上清，空气中干燥沉淀5～10min；(10)加入10μL RNase-free水溶解，测浓度后-80℃冰箱保存。
3、转录组测序实验步骤：提取样品总RNA并使用DNase I消化DNA后，用带有Oligo(dT)的磁珠富集真核生物mRNA；加入打断试剂在Thermomixer中适温将mRNA打断成短片段，以打断后的mRNA为模板合成一链cDNA，然后配制二链合成反应体系合成二链cDNA，并使用试剂盒纯化回收、粘性末端修复、cDNA的3’末端加上碱基“A”并连接接头，然后进行片段大小选择，最后进行PCR扩增；构建好的文库用Agilent 2100Bioanalyzer和ABI StepOnePlus Real-Time PCR System质检合格后，使用Illumina HiSeqTM 2000或其他测序仪进行测序。
4、信息分析流程：由Illumina HiSeqTM 2000测序所得的数据称为raw reads或raw data，随后要对raw reads进行质控(QC)，以确定测序数据是否适用于后续分析。质控后，经过滤得到clean reads，用SOAPaligner/SOAP2[16]将clean reads比对到参考序列。在比对后，以reads在参考序列上的分布情况及覆盖度，来作为判断比对结果是否通过第二次质控的指标。如果通过第二次质控，则进行基因表达 等后续分析。
5、转录组数据分析：基因表达量的计算使用RPKM[17]法(Reads per kilobase transcriptome per million mapped reads)，其计算公式为：
Figure PCTCN2017089556-appb-000001
公式中假设RPKM(X)为基因X的表达量，那么C是唯一比对到基因X的reads数，N是唯一比对到参考基因的reads总数，L为基因X编码区的碱基数。RPKM法作为一个标准化处理，能消除测序量差异和基因长度对计算基因表达的影响，所计算得到的基因表达量可以用于直接比较不同样品间的基因差异表达。
以上的检测结果见图2，结果表明三个品种间的基因表达出现了明显的与产肉量相符的梯度差异性规律。促进成肌的基因或者miRNA的起始表达一般在五指山最早最多，而长白猪表达时间较晚，如MyoD、MyoG、miR-133b等。而抑制成肌的基因则在胚胎发育早期在长白猪中表达最多，其次是蓝塘猪、五指山猪，如ID2、MDFI和MSTN(28dpc)。肌管分化的标志基因在五指山猪的表达也最早、最多，长白猪则出现最晚、最少，如MYH3、MYH13、DES等，这一现象也与之前组织形态学观察的结果吻合。
实施例3五指山与长白猪MyoD表达情况的定量qPCR与western blot检测。
1、样品采集：同实例一样品采集部分。
2、胚胎RNA提取：同实例2胚胎RNA提取步骤。
3、RNA逆转录得到cDNA(具体方法参考本领域常规技术操作或各种商业化试剂盒的说明书)，cDNA置于-20℃，使用时稀释5倍。
4、实时荧光定量PCR：反应体系如表1：先将水加入cDNA中混合，再加到384孔板中，随后将引物与
Figure PCTCN2017089556-appb-000002
qPCR Mix混匀，加入对应的孔中，GAPDH作为内参基因，数据分析使用高级相对定量2-ΔΔCt，定量引物见表2。将表1反应体系混匀后加入384孔板中，3000转离心2min；在LightCycler480Ⅱ上进行荧光定量PCR，反应程序为：预变性95℃，10min；变性95℃，5s；退火60℃，1min；延伸72℃，30s；40个循环。分析溶解曲线：95℃，5s；65℃，15s；95℃，0s。
表1为qPCR的反应体系
Figure PCTCN2017089556-appb-000003
表2为qPCR引物序列
基因 上游引物(5'to 3') 下游引物(5'to 3')
GAPDH GCCTCCAAGGAGTAAGAAAC GAAATTGTGAGGGAGATGCT
MyoD ACCGCTCCGCGACGTAGATT GCGAGTGTTCCTCGGGCTTT
5、Western blot检测，具体步骤如下：(1)组织蛋白的提取：将少量组织块置于加入磁珠的组织破碎管中，加入前用剪刀剪碎，加入400μL含有PMSF的蛋白裂解液于组织破碎管中，在组织破碎仪上进行匀浆，然后置于冰上，重复几次使组织尽量碾碎，裂解30min；将裂解液移至1.5mL离心管中，并于12,000rpm4℃下离心5min，随后用0.5mL离心管分装上清液并置于-20℃保存；(2)蛋白定量：使用考马斯亮蓝法对蛋白样品浓度进行均一。首先稀释5×考马斯亮蓝至1×，酶标板每孔加入180μL的考马斯亮蓝液，每个样品重复3次。在室温下震荡30s混匀，静置5min后用595nm的波长测吸光值，绘制标准曲线计算蛋白浓度；(3)蛋白样品电泳：在放蛋白样品的离心管中加入5×蛋白上样缓冲液，干式恒温仪100℃加热10min变性蛋白质。每个孔加入10～30μg蛋白样品(根据蛋白浓度和表达情况确定)，电泳条件：80V，45min；120V，80min；(4)转膜：半干转，首先在转膜缓冲液中浸润剪好的滤纸，随后将PVDF膜放入甲醇活化1min，阳极板从上到下依次放入滤纸、SDS-PAGE胶、PVDF膜、滤纸(每层均不要有气泡)，盖上阴极板，转膜条件：20V，60min；(5)封闭：转膜后将PVDF 膜放入封闭液(TBST配制的5％脱脂牛奶)室温震荡封闭1小时或4℃冷库过夜；(6)一抗孵育：将膜放入稀释好的一抗中，4℃冷库过夜，之后在摇床上用TBST洗3次，每次10min；(7)二抗孵育：将膜放入稀释好的二抗(1:5000)中，室温孵育1h，之后再摇床上用TBST洗3次(每次10min)；(8)用滤纸吸干膜上的水，然后覆盖ECL显色液避光1min后用ECL显色仪拍照。
以上的检测结果见图3，结果显示，胚胎期18～28天三品种猪均没有肌纤维形态产生，到了胚胎32天，五指山猪首先出现了中心圆环状的初级肌纤维；胚胎35天时，蓝塘和长白猪均出现了初级肌纤维，但前者数量明显多于后者，而五指山猪的初级肌纤维继续增多和直径加大；到了胚胎42天，三品种猪初级肌纤维继续分化。
实施例4 C2C12细胞中过表达MyoD抑制增殖
1、转染：(1)转染前一天将C2C12细胞接种于6孔板，密度约为30％～50％，培养于完全培养基；(2)转染试剂配制：用250ul OPti-MEM稀释5ul的miRmimic或是10ul inhibitor及相应的对照miRNC；另250ul OPti-MEM稀释5ul lipofectamine2000TM，于室温下放置5分钟；(3)将(2)中的二者轻轻混匀于室温孵育15分钟，形成转染复合物；(4)将(3)中的转染复合物加到细胞中，再加入培养基，轻轻混匀，6小时后更换新鲜培养基。
2、实时无标记细胞检测：利用实时无标记细胞分析仪(Real-Time Cell Analyzer)分析细胞的增殖。仪器调试校准：预热仪器，然后增殖检测板(E-Plate16)每孔中加50μL含10％FBS的DMEM，装回仪器，调整基线。细胞增殖检测：将处理过的细胞消化计数加入增殖检测板中，每孔加入8000个细胞，最终用10％FBS的DMEM将每孔的培养基补齐180ul，每组实验3个重复。设置5min测试一次，测试时长72h后绘制细胞增殖曲线。
3、EDU增殖检测：(1)EDU用10％的DMEM培养基按1:1000的比例稀释，12孔板每孔加入300ul，细胞培养箱中孵育2h；(2)每孔用500ul的PBS洗2次，5min/次；(3)每孔用500ul 4％的多聚甲醛室温固定10min；(4)PBS洗2次，5min/次；(5)每孔用500ml 0.5％的tritonX-100室温孵育10min；(6)重复步骤(4)；(7)300ul/孔Apollo染色反应液(根据说明配制)，室温避光孵育30min；(8)PBS洗1次，5min；(9)室温下，每孔300ul DAPI染色5min；(10)PBS清洗3次，5min/次；(11)倒置荧光显微镜下拍照。
4、PI细胞周期检测：(1)前一天，把细胞消化下来装于1.5ml EP管中，用PBS清洗，去除培养基和胰酶；(2)向每管中加入1ml 70％预冷的乙醇，4℃固定过夜；(3)1500r/s离心5分钟收集细胞去除乙醇，用PBS洗3次，每次5分钟；(4)每孔加入1ml PI工作液，室温避光孵育30min。(5)过滤上机检测。
5、实时荧光定量PCR:(1)细胞总RNA提取：参考本领域常用的TRIzol提取法或者使用商业化的提取试剂盒；(2)RNA反转为cDNA：参考实施例3；(3)SYBR GreenΙReal-time PCR：参考实施例3。
5、载体构建：(1)质粒DNA提取：参照Genstar质粒提取试剂盒说明书。
(2)目的基因及线性化载体的获得：A:PCR：反应体系见表3，模板为C2C12细胞DNA。反应程序为：预变性95℃，30s；变性95℃，10s；退火、延伸68℃，2min(根据基因大小和酶的扩增速率)；延伸72℃，5min；39个循环。B:合成片段退火:化学合成的单链DNA需要退火成为双链，反应体系见表4。退火程序为95℃，10min；85℃，1min；75℃，1min；65℃，1min；55℃，1min；45℃，1min；30℃，1min。C:载体双酶切和连接：利用限制性核算内切酶将载体双酶切使其线性化，酶切反应体系参照表5，37℃水浴酶切4小时后琼脂糖凝胶回收，将获取的线性化载体和基因片段按表6体系配制后16℃水浴连接过夜。D:转化和鉴定：(1)从-80℃冰箱取出DH5α感受态细胞，冰上融化后在超净台内加入连接产物，轻轻震荡使其混匀，冰浴30min；(2)42℃热激45s后立即冰浴2min；(3)每管加入600μL无抗LB液体培养基，37℃、220rpm震荡培养30min；(4)3000×g、1min离心弃上清浓缩菌体，将菌体均匀涂布于有相应抗生素的LB固体培养基平板上，37℃倒置培养12-16小时；(5)挑取单克隆菌落接种于600μL含抗生素的LB液体培养基中，37℃、220rpm震荡培养5小时，菌液PCR并经测序鉴定阳性克隆。E:Western blot实验：(1)样品处理：用PBS清洗2遍细胞，吸干残留液体后加入150～300μl含PMSF的蛋白裂解液，在冰上充分搅拌至细胞完全裂解，转移至1.5ml离心管中并用1ml注射器反复吹打至样品不再粘稠。(2)其余Western blot操作步骤参考见实施例3相关内容。F:数据处理及统计分析：除特别说明，每个生物学实验至少重复3次，实验结果用品均值±标准误差(Mean±SEM)表示；实验作图使用Prism 6软件，数据统计分析使用SPSS(version 21)， 两组数据间的统计分析均采用双侧检验，三组以上数据间的统计分析采用ANOVA，并表示为：显著性差异*：p<0.05；极显著差异**：p<0.01；***：p<0.001。
表3为PCR反应体系
组分 体积(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
表4为单链退火反应体系
组分 体积(μL)
Forward Fragment 2
Reverse Fragment 2
10×T4 ligase Buffer 2
T4 PNK 1
H2O Add to 20uL
表5为双酶切体系
组分 体积(μL)
Plasmid DNA 2ug
10×fermentas Buffer 5
Enzyme 1 1
Enzyme 2 1
H2O Add to 50
表6为连接反应体系
组分 体积(μL)
10×T4 ligase buffer 1
Vector Fragment 20ng
DNA Fragment 80ng
T4 ligase 0.3
H2O Add to 10
以上检测结果见图4，结果显示过表达MyoD会抑制C2C12细胞的增殖。
实例例5 C2C12细胞增殖期越早过表达MyoD则分化后产生的肌管越少
1、诱导分化：待细胞完全长满，弃培养基，换成含2％马血清的DMEM培养基培养，每两天更换一次培养基。
2、免疫荧光：(1)细胞用4％对聚甲醛室温固定10min；(2)0.5％的tritonX-100室温孵育10min；(3)用3％BSA封闭30min；(4)用3％BSA稀释的一抗在4℃过夜孵育；(5)吸去一抗，用PBS洗涤3个5min；(6)用相应的荧光二抗室温避光孵育1h；(7)吸去二抗，用PBS洗涤3个5min；(8)DAPI染色5min；(9)PBS洗3个5min；(10)荧光显微镜下拍照。
3、Western blot和实时荧光定量PCR：参考实施例4。以上检测结果见图5，结果显示在C2C12细胞增殖阶段过表达MyoD会抑制成肌分化过程，越早过表达MyoD，产生的肌管越少。
实施例6 NIH3T3细胞中过表达MyoD会抑制增殖
1、诱导分化：参考实施例5相关部分。
2、免疫荧光：参考实施例5相关部分。
3、Western blot和实时荧光定量PCR：参考实施例4相关部分。
4、实时无标记细胞检测、EDU染色和载体构建：参考实施例4相关部分。
以上检测结果见图6和图7。

Claims (4)

  1. 推迟猪胚胎期成肌分化因子MyoD的表达时间在提高猪产肉量中的应用。
  2. 一种通过延迟基因的表达时间来提高猪瘦肉产量的方法，其特征在于，推迟猪胚胎期成肌分化因子MyoD的表达时间，从而提高猪产肉量。
  3. 根据权利要求2所述的方法，其特征在于，推迟MyoD基因在猪中表达1周以达到增加产肉量的效果。
  4. 根据权利要求2所述的方法，其特征在于，所述推迟猪胚胎期成肌分化因子MyoD的表达时间的的方法如下：在地方猪种中利用dox诱导的Cre-LoxP敲入MyoD基因的技术，通过Cre-LoxP猪与CRISPR-Cas9技术构建的MyoD敲入猪杂交得到的MyoD-LacZ-Stop-Cas9-KI猪；或在不同的胚胎时期对母猪饲喂dox，诱导MyoD基因表达的时序，使地方猪种MyoD基因表达时间推迟；或利用慢病毒介导的RNA干扰技术构建MyoD基因的慢病毒干扰载体，在不同胚胎时期对地方猪种的母猪注射慢病毒干扰载体从而推迟MyoD的表达时间。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997023644A1 (en) * 1995-12-22 1997-07-03 Cofok B.V. The pig myogenin gene and method to identify polymorphisms related to muscle growth
CN104388465A (zh) * 2014-09-24 2015-03-04 华南农业大学 Mdfi在调控猪骨骼肌生长发育中的应用
CN106535630A (zh) * 2014-04-28 2017-03-22 重组股份有限公司 猪中的多重基因编辑

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997023644A1 (en) * 1995-12-22 1997-07-03 Cofok B.V. The pig myogenin gene and method to identify polymorphisms related to muscle growth
CN106535630A (zh) * 2014-04-28 2017-03-22 重组股份有限公司 猪中的多重基因编辑
CN104388465A (zh) * 2014-09-24 2015-03-04 华南农业大学 Mdfi在调控猪骨骼肌生长发育中的应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
COUTINHO, L.L. ET AL.: "Delayed Somite Formation in a Quail Line Exhibiting Myofiber Hyperplasia is Accompanied by Delayed Expression of Myogenic Regulatory Factors and Myosin Heavy Chain", DEVELOPMENT, vol. 117, no. 2, 1 February 1993 (1993-02-01), pages 563 - 569, XP055527804, ISSN: 1477-9129 *
TE PAS, M.F.W. ET AL.: "Influences of Myogenin Genotypes on Birth Weight, Growth Rate, Carcass Weight, Backfat Thickness, and Lean Weight of Pigs", JOURNAL OF ANIMAL SCIENCE, vol. 77, no. 9, 30 September 1999 (1999-09-30), pages 2352 - 2356, XP055527805, ISSN: 0021-8812 *
TE PAS, M.F.W. ET AL.: "Transcriptome Expression Profiles in Prenatal Pigs in Relation to Myogenesis", JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY, vol. 26, no. 2-3, 1 July 2005 (2005-07-01), pages 157 - 165, XP019256279, ISSN: 1573-2657 *

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