CN109337926B - Method for constructing haplotype chicken myoblasts with high let-7b mature body expression - Google Patents

Abstract

The invention discloses a method for constructing haplotype chicken myoblasts with high let-7b mature body expression quantity, which comprises the following steps: 1) cloning a haplotype expression vector; 2) culturing primary myoblasts of chickens; 3) and (4) cell transfection. By using

Description

Method for constructing haplotype chicken myoblasts with high let-7b mature body expression

Technical Field

The invention relates to the field of biological gene engineering, in particular to a construction method of a haplotype chicken myoblast.

Background

MicroRNA (miRNA) is an endogenous non-coding single-stranded RNA with about 22 nucleotides. With the rapid development of molecular biology research techniques and methods, researchers have found more and more mirnas. The miRNA is generated by shearing a miRNA precursor (pre-miRNA) which is 70-80 nucleotides in length and comprises a hairpin structure. MiRNA regulates genes by complementing a3 'non-coding region (3' UTR) of mRNA of its target gene, so that translation of the mRNA is inhibited. Although mirnas do not directly encode proteins, the RNA that they encode plays an important role throughout the life of an organism. miRNAs are now found to be involved in a number of regulatory pathways including skeletal muscle development, viral defense mechanisms, hematopoietic processes, organogenic differentiation, cell proliferation and death, fat metabolism, etc.

In recent years, with the rapid development of technologies such as genomics and genome-wide association analysis, mirnas have become popular fields in animal genetic breeding research at home and abroad. In China, Goldine and the like perform miRNA sequencing on mammary tissues of high-milk-quality cows (H) in the lactation period and low-milk-quality cows (L) in the lactation period of Holstein cows by utilizing a high-throughput sequencing technology, and differential miRNAs between groups are screened by differential expression analysis to obtain 56 differential expression miRNAs (P <0.05), and target gene prediction is performed on the differential expression miRNAs, and 4 functional genes which are reported to be closely related to milk protein and milk fat are discovered by screening the target genes: CSN3, SCD, LALBA, and DGAT 2. The biological functions of target gene aggregation are mostly involved in protein and fat metabolism, mammary gland development and differentiation, and immune functions. Solexa sequencing and bioinformatics analysis of goat muscle tissue by the Lingyui et al identified 517 species-conserved and 2 goat genome-specific miRNAs. In the earlier stage of the subject group, a target prediction software is used for predicting and a dual-luciferase target verification system is used for verifying that GHR and IGF2BP3 are target sites of let-7b, the overexpression of the let-7b can cause the obvious down-regulation of the expression quantity of mRNA of the GHR and IGF2BP3, the let-7b is proved to have a negative regulation effect on GHR and IGF2BP3 genes, and meanwhile, the let-7b is found to influence a JAK-STAT signal channel through mediating the GHR gene, so that the growth and development of skeletal muscles are regulated. There are also many reports that miRNAs are widely involved in a series of vital activities such as growth and development of chicken embryos, sexual maturation, proliferation and differentiation of nerve cells and muscle cells, and cell migration [29-33 ].

Single Nucleotide Polymorphisms (SNPs) refer to mutations that occur in a Single Nucleotide in the genomic sequence of a living individual. SNPs occur at the DNA level with a very high frequency, up to nine or more in all mutant forms, but many of them are concentrated in non-coding regions, and few others are distributed in coding regions of genes. There is evidence that SNP can affect the expression and function of RNA, DNA and protein levels, and is an important factor causing the occurrence of diseases, drug sensitivity and phenotype differences between individuals and groups, and SNP on the mature body and precursor sequence changes its formation process, even changes the target site of the mature body or changes the binding strength with the target gene, so that the function of miRNA changes, and thus animal traits change. Each miRNA has more than one target site, which means that SNP positioned on the miRNA has important significance, so that the SNPs on the mature body sequence and the precursor sequence of the miRNA are screened to reveal the functions of the SNPs, which is of great research significance. Currently, the search and screening of SNPs is performed by using sanger sequencing or PCR-RFLP method, and due to the low efficiency of these technical methods, the genechip technology and High-throughput sequencing which have been rapidly developed in recent years can perform efficient screening of mutation sites on the genome level. Research shows that SNP located in a miRNA seed region is more scarce, and that a large number of SNP located in a miRNA precursor region is widely applied to the fields of genetic diagnosis, tumor susceptibility and the like.

The genetic polymorphism of mirnas can affect pre-and post-transcriptional regulation by altering the target site of mirnas and altering the stability of mrnas, which in turn affect gene expression and phenotypic differences. These polymorphic sites are expected to be useful as molecular markers for auxiliary selection in breeding. In the study of 313 RXRG gene mutation only located in sheep exon 2 and the relation between the RXRG gene mutation and twins characters, the gene polymorphism of the 1 st exon, the 2 nd exon and the 10 th exon of the RXRG gene is analyzed by PCR-SSCP, and the result shows that the genotypes of 3P 2 fragments are obviously related to the twins characters in a population (P is less than 0.05). Lanxian warong, etc. through 45 parts of inner Mongolia white cashmere goat alpha-lactalbumin (LALBA) gene to carry out sequencing and typing, and carry out correlation analysis with cashmere quantity, cashmere thickness, cashmere length and weight characters. The results showed that 1 mutation site g.1897t > C was present in the region of exon 3. Different genotypes of the locus are obviously related to the down production (P is less than 0.05), and the down production of TC genotype individuals is higher than that of TT genotype individuals by 142.68g (up to 26.21 percent) (P is less than 0.05). Lei et al (2010) experiments demonstrated that the T/C mutation on the miR27a gene is associated with litter size in pigs. Huang used PCR restriction fragment length polymorphism and micro-sequencing methods to perform SNP genotyping on 187 ducks. The hatchability of the CC genotype and the TT genotype is 79.59 +/-3.40 and 76.35 +/-1.77 respectively, which is obviously higher than that of the CT genotype (65.77 +/-2.07) (P < 0.05).

The let-7 family is a class of miRNAs discovered by Reinhart et al at the beginning of this century. let-7 family members are located on several different chromosomes, respectively. Let-7b, one of the let-7 family members, was found to play an important role in the targeting of regulated genes in many signaling pathways, many of which are associated with proliferative apoptosis of cells, and thus there are many researchers investigating the relationship between let-7b and cell proliferation. Studies by Sch. mu. Ltz, et al, found that let-7b inhibited cell cycle progression and anchorage-independent growth of melanoma cells. Zhao et al found in the study that let-7b could regulate the proliferation and differentiation of neural stem cells by targeting stem cell regulator TLX and cell cycle regulator cyclinD 1; the knockout promotes the proliferation of neural stem cells and promotes neural differentiation; and over-expression of let-7b can reduce neural stem cell proliferation. In zebra fish experiments, the Chennan shows that the inhibition of hypoxia-inducible factor 1(hif-1a) can cause the expression of let-7b to be reduced, the overexpression of let-7b in ZF4 cells can change the cell cycle process, block the transition from the G1 stage to the S stage in the cell cycle, and stop or slow down the proliferation of the cells; it was also identified by in vitro experiments that the target gene foxh1 of let-7b is also directly or indirectly involved in the cell proliferation process. The results of a scarification experiment performed after miRlet-7b and anti-miRlet-7b are transfected into breast cancer MCF-7 cells show that let-7b can remarkably inhibit the migration capacity of breast cancer MCF-7 cells, while an anti-miR let-7b group can remarkably promote the migration capacity of breast cancer MCF-7 cells, and prove that let-7b can inhibit the migration capacity of breast cancer MCF-7 cells.

The SNP site in the precursor region gene sequence of the chicken let-7b is associated with economic traits to obtain that the SNP site is obviously or extremely obviously related to important traits such as leg muscle weight, leg muscle crude protein content and the like, and the SNP site is preliminarily presumed to influence the expression quantity of a mature body by influencing the processing process of miRNA, thereby influencing a series of regulation and control expression processes.

Disclosure of Invention

The invention aims to construct haplotype recombinant plasmids with different SNP combinations, transfect to primary chicken myoblasts and determine the influence of different haplotypes on the expression quantity of a let-7b mature body, thereby obtaining the haplotype chicken myoblasts with high expression quantity of the let-7b mature body.

The technical scheme adopted by the invention is as follows: a method for constructing haplotype chicken myoblasts with high let-7b mature body expression quantity comprises the following steps:

1) haplotype expression vector cloning

a. Designing a primer pair according to the sequencing result of a let-7b precursor region gene sequence, wherein a target fragment comprises g.270G > A, g.526G > A, g.678T > C and g.710G > C4 mutation sites, and simultaneously carrying out haplotype gene PCR amplification after adding an enzyme digestion site and a protective base at the 5' end of the designed primer pair to obtain a PCR product;

b. recovering and purifying the PCR product to obtain purified DNA solution containing the target segment for later use;

c. adopting pcDNA3.1 strain to carry out bacterial liquid propagation, extracting pcDNA3.1 circular plasmid and obtaining a purified DNA sample;

d. b, performing double enzyme digestion treatment on the DNA solution containing the target fragment obtained in the step b and the DNA sample obtained in the step c by using endonuclease respectively, and purifying double enzyme digestion products to obtain a target gene fragment and a pcDNA3.1(+) plasmid vector;

e. d, connecting the target gene fragment obtained in the step d with pcDNA3.1(+) plasmid vectors by using T4 DNA Ligase, converting the connection products, and performing shake propagation to obtain 5 successfully constructed haplotype recombinant plasmids;

2) culture of primary myoblasts in chickens

Separating myoblasts from the eggs of the chickens which are hatched for 11d, and purifying for later use;

3) cell transfection

By using

The reagent method respectively transfects 5 haplotype recombinant plasmids to the primary myoblasts of the chicken to obtain 5 haplotype myoblasts with high let-7b mature body expression quantity.

The invention has the beneficial effects that: the invention constructs different haplotype over-expression vectors, and the vectors are transfected into primary chicken myoblasts to determine the influence of different haplotypes on the expression quantity of a let-7b mature body, so that the haplotype chicken myoblasts with high let-7b mature body expression quantity are obtained, and the reason for the difference of the expression quantity is inferred to be that the difference of the expression quantity of the let-7b mature body is caused because different SNPs on a let-7b precursor region influence the processing process of the let-7b precursor.

Drawings

FIG. 1 shows the results of the double digestion in FIG. 2.6 (A is a double digestion electrophoretogram of pcDNA3.1; B is a double digestion electrophoretogram of different haplotypes);

FIG. 2 is a partial bacteria liquid identification electrophoresis chart in FIG. 2.11;

FIG. 3 shows the sequencing result of the bacterial liquid in FIG. 2.11 (A is the upstream restriction enzyme site in the sequencing result of the bacterial liquid, B is the downstream restriction enzyme site in the sequencing result of the bacterial liquid);

FIG. 4 shows the situation of 4 mutation sites introduced in 2.11 (A is g.270A > G mutation site in bacterial liquid sequencing, B is g.526A > G mutation site in bacterial liquid sequencing, C is g.678T > C mutation site in bacterial liquid sequencing, and D is g.710G > C mutation site in bacterial liquid sequencing);

FIG. 5 shows the fluorescence (X40) of primary myoblasts from chicken in 3.2 (A is DAPT staining; B is Desmin fluorescent protein staining);

FIG. 6 shows the relative expression levels of let-7b transfected with different haplotypes in 3.9 (where the vertical axis 1 represents the AGCC haplotype; 2 represents the AACC haplotype; 3 represents the GATC haplotype; 4 represents the GACG haplotype; 5 represents the GACC haplotype; and 6 represents pcDNA3.1 empty vector).

Detailed Description

The present invention is specifically described below with reference to examples in order to facilitate understanding of the present invention by those skilled in the art. It should be particularly noted that the examples are given solely for the purpose of illustration and are not to be construed as limitations on the scope of the invention, as non-essential improvements and modifications to the invention may occur to those skilled in the art, which fall within the scope of the invention as defined by the appended claims. Meanwhile, the raw materials mentioned below are not specified in detail and are all commercial products; the process steps or preparation methods not mentioned in detail are all process steps or preparation methods known to the person skilled in the art.

First, experimental material

1.1 Experimental animals

Hatching eggs isolated into myocytes were purchased from the chicken farm of southern agricultural university, south China.

1.2 Main reagents and preparation solutions

DNA purification recovery kit was purchased from OMEG corporation; trizol and RNA solid phase scavenger were purchased from Invitrogen; plasmid extraction kits were purchased from OMEG corporation; SYBR Green PCR Master Mix quantification kit was purchased from TOYOBO, Japan; the miRNA qRT-PCR reverse transcription kit is purchased from Ruibo Biotechnology limited, Guangzhou; 1640 culture medium, fetal bovine serum, anti-Desmin,

Purchased from semer feishel corporation; PBS was purchased from Dingguo Bio Inc. Restriction enzymes were purchased from seimer feishel technologies ltd; DH5 alpha, pcDNA3.1 vector genes were purchased from TaKaRa Bio Inc., Dalian; agar powder, yeast extract, peptone, ampicillin, Tris alkali, chloroform, absolute alcohol, isopropanol, sodium chloride and the like are all made in China or imported analytical purifications. Preparation of other reagents:

LB liquid medium: 4g Tryptone, 2g Yeast Extract, 4g NaCl, double distilled Water (ddH)₂O) constant volume to 400mL, autoclaving and storing at 4 ℃.

LA liquid medium: 400 mu L of ampicillin is added into 400mL of LB liquid culture medium stored at 4 ℃, thus obtaining LA liquid culture medium.

Ampicillin-containing solid LA medium: 4g Tryptone, 2g Yeast Extract, 4g NaCl, 10g agar powder, double distilled water (dd)H₂O) constant volume to 400mL, and autoclaving. Adding ampicillin when it is cooled to a temperature of not scalding hands, mixing completely, subpackaging into sterilized culture dish, and storing at 4 deg.C after it is solidified.

Ampicillin stock solution: ampicillin was prepared at 100mg/mL with double distilled water, which was sterilized by filtration through a 0.22mm filter and stored at-20 ℃ at a working concentration of 100 mg/mL.

Cloning of two, different haplotype expression vectors

2.1 primer design

According to the sequencing result of the gene sequence of the let-7b precursor region, Primer premier5.0 software is used for designing a Primer pair, a target fragment is required to contain g.270G > A, g.526G > A, g.678T > C and g.710G > C4 mutation sites, a proper enzyme cutting site and a protective base are added to the 5' end of the designed Primer, the information of the Primer pair is shown in Table 1, wherein the bold part is a protective base sequence, the underlined parts are enzyme cutting sites (EcoR I and Xba I), and the sequences of an upstream Primer and a downstream Primer in the Primer pair are respectively shown in SEQ ID NO1 and SEQ ID NO 2.

TABLE 1 primer pair information

2.2 haplotype Gene amplification

The PCR amplification reaction system was prepared according to the following Table 2, and the PCR reaction procedure was: pre-denaturation at 94 deg.C for 2min, 40 cycles (denaturation at 98 deg.C for 10s, denaturation at 54 deg.C for 20s, and extension at 72 deg.C for 2min30s), extension at 72 deg.C for 10min, and storage at 4 deg.C. The PCR product was checked by electrophoresis on 1.5% agarose gel, and the product was checked for eligibility for further experiments.

TABLE 2 let-7b precursor region PCR reaction System

2.3 recovery and purification of PCR products

And (4) performing gel cutting recovery and purification on the PCR product qualified in the electrophoresis detection. The procedure was performed according to the instructions of the Hipure Gel pure DNA Mini Kit (magenta) agarose Gel DNA recovery Kit to obtain a purified DNA solution containing the desired fragment for use.

2.4 propagation of bacterial liquid

The pcDNA3.1 strain is a strain preserved in the laboratory. The liquid transfer gun sucks 200 mu L of strain and inoculates the strain into 20mL LA liquid culture medium, and the strain is shaken by a shaking table at 200rpm and 37 ℃ for 8h, and then the extracted vector plasmid is taken out.

2.5 pcDNA3.1 circular plasmid extraction

(1) Taking out the bacterial liquid at the room temperature of 3000-;

(2) adding 500 mu L of Buffer E1 with RNase A into the cell sediment, performing high-speed vortex oscillation for 15s to resuspend the bacterial liquid, and transferring to a new 2mL centrifuge tube;

(3) add 500. mu.L of Buffer E2 to the cell suspension, mix gently and reverse the tube 8-10 times, do not shake, the mixture becomes viscous and transparent. Standing at room temperature for 2 min;

(4) adding 500 mu L of precooled Buffer E3, immediately and slightly reversing the centrifuge tube from top to bottom for 10-15 times, uniformly mixing to generate white precipitate, and centrifuging for 10min at 13000 Xg without shaking (the step is a key step of plasmid yield);

(5) sleeving a Hipure EF Mini Column on a collecting tube, transferring 750 mu L of supernatant into a Column, adding 0.1-time volume of ETR Solution, reversing and mixing uniformly for several times, incubating for 10min (reversing for several times in the period), and gradually clarifying the liquid;

(6) followed by a water bath for 5min, at which time the tube became cloudy again. Centrifuging at 12000 Xg for 3 min;

(7) transferring the supernatant into another clean 1.5mL tube, adding 0.5-time volume of absolute ethyl alcohol, slightly reversing for 6-7 times, uniformly mixing, and standing at room temperature for 1-2 min;

(8) transferring the mixture of the last step into a clean HiBindTM DNAmi Columus adsorption column, and centrifuging at 10000 Xg for 1 min;

(9) discarding the filtrate, and continuously transferring the liquid in the step (7) until all the liquid passes through the column;

(10) adding 500 μ L Buffer HB into the adsorption column, centrifuging at 10000 × g for 1 min;

(11) adding 700. mu.L of DNA Washing Buffer into the adsorption column, and centrifuging for 1min at 10000 Xg;

(12) repeating (11) once;

(13) discarding the filtrate, and centrifuging for 2min by an empty column of 13000 Xg;

(14) placing the adsorption column in a new 1.5mL centrifuge tube, adding 45 μ L Buffer TE to dissolve the plasmid;

(15) purified DNA samples were obtained and stored at-20 ℃.

2.6 double digestion

Carrying out double enzyme digestion treatment on the DNA solution containing the target fragment obtained in the step 2.3 and the DNA sample obtained in the step 2.5 respectively by using endonucleases EcoR I and Xba I, wherein an enzyme digestion system is shown in the following table 3, the DNA solution is subjected to enzyme digestion for 2h in an incubator at 37 ℃, the double enzyme digestion result is shown in a figure 1A and a figure 1B, electrophoresis patterns of the figure 1A and the figure 1B are obtained by carrying out double enzyme digestion experiments on amplification products of different haplotypes with enzyme digestion sites and pcDNA3.1 plasmid, and the size of the product accords with the expected product length and can be used for subsequent experiments.

TABLE 3 double enzyme digestion System

2.7 purification of the double digestion product

Adopting an OMEGA DNA purification recovery kit to recover a PCR product, wherein the PCR product recovery method comprises the following steps:

(1) performing instantaneous centrifugation on the PCR product, measuring the volume of the product by using a pipette gun, and transferring the product into a centrifuge tube of 1.5/2.0 mL;

(2) adding equivalent volume of Buffer GDP, and mixing by reversing or whirling;

(3) sleeving a Hipure DNA column in a collecting pipe, and transferring the mixed solution into the column for 1000 Xg centrifugation for 30-60 s;

(4) discarding the filtrate, putting the column back into the collection tube again, adding 600 μ L Buffer DW2 (diluted with absolute alcohol, DW 2: absolute alcohol 1:4) into the column, and centrifuging at 12000rpm for 30-60 s;

(5) discarding the filtrate, putting the column back into the collection tube again, adding 600 μ L Buffer DW2 (diluted with absolute alcohol, DW 2: absolute alcohol 1:4) into the column, and centrifuging at 12000rpm for 30-60 s;

(6) discarding the filtrate, replacing the column with the other, and centrifuging at 12000rpm for 2 min;

(7) the column was mounted in a new 1.5mL centrifuge tube, 45. mu.L of an Elution Buffer preheated at 55 ℃ was added to the center of the column membrane, left to stand at room temperature for 2min, and centrifuged at 12000rpm for 1min to elute the DNA;

(8) detecting the purified product double enzyme digestion product by electrophoresis, and storing at-20 ℃ for later use.

2.8 connection

The purified target gene fragments were ligated with pcDNA3.1(+) plasmid vectors using T4 DNA Ligase, respectively, and the ligations were performed overnight at 16 ℃ by the PCR system as shown in Table 4 below.

TABLE 4T 4 DNA ligase ligation System

2.9 transformation

The ligation products were transformed by first formulating the transformation system in a clean bench, the system components being shown in table 5 below.

TABLE 5 ligation product conversion System

The transformation method comprises the following steps: the transformation system is ice-bathed in an ice box for 25min, then heat-shocked in an ice chain instrument at 42 ℃ for 1min, placed in the ice box for ice-bathing for 2min, and then placed in a clean bench, the transformation system and 500 mu L of LB without ampicillin resistance are mixed evenly and shaken in a shaking table at 37 ℃ and 200rpm for 1h, after the shaking is finished, the transformation system is centrifuged at 3000rpm for 4min, 300 mu L of supernatant is discarded, the remaining mixture is lightly resuspended, and the mixture is coated on a LA plate containing ampicillin in a constant temperature incubator at 37 ℃ for overnight culture.

2.10 shaking bacteria

When the strain grows out of the LA plate, a single colony is picked in a super clean bench to a 1.5mL centrifuge tube added with 800 μ L LA liquid culture medium, and the centrifuge tube is placed in a shaker at 37 ℃ and 200rpm for 4h for propagation.

2.11 Positive colonies PCR and sequencing identification

And carrying out bacteria liquid PCR and sequencing on the amplified bacteria liquid so as to detect whether the target fragment is successfully connected with the pcDNA3.1 vector. The PCR system of the bacterial liquid is shown in the following table 6, and the PCR reaction procedure is as follows: pre-denaturation at 94 deg.C for 5min, 38 cycles (denaturation at 98 deg.C for 30s, annealing at 56 deg.C for 30s, and extension at 72 deg.C for 30s), extension at 72 deg.C for 7min, and storage at 4 deg.C.

TABLE 6 bacterial liquid PCR System

Carrying out 1.5% agarose gel electrophoresis detection on a PCR product of the bacterial liquid, carrying out PCR bacterial liquid identification on the PCR product by using a universal primer T7, wherein the identification result is shown in figure 2, a single band in an electrophoretogram is a successfully-constructed haplotype expression vector, a vector band containing a target fragment is about 1000bp, a bacterial sample with the consistent band size is sent to a biotechnology limited company for sequencing, the sequencing result is shown in figures 3A and 3B, the sequencing result shows that the enzyme cutting site is added for PCR amplification, the enzyme cutting site is successfully introduced into a target sequence, the left side is a pcDNA3.1 vector sequence, the right side is a different haplotype target sequence according to the comparison of sequences at two wings of the enzyme cutting site in figure 3A, the left side is a different haplotype target sequence, and the right side is a pcDNA3.1 vector sequence, which shows that the sequences at two ends are successfully connected. The situation of the introduced 4 mutation sites is shown in FIGS. 4A, 4B, 4C and 4D, respectively, which indicates that the vector construction is successful. The sequencing results of the open cell suspension were obtained for all haplotypes, 5 in total, using the Seq-man software, as shown in Table 7 below. And (4) performing propagation and seed preservation on the bacteria sample with correct sequencing for subsequent experiments.

TABLE 7 let-7b precursor haplotypes

Culture of primary myoblasts

3.1 isolation and purification of myoblasts

(1) Taking out the hatching eggs which are hatched for 11d from the incubator, and wiping the eggshells by 75 percent for disinfection. Gently breaking the hatching egg air chamber by using the big head of the tweezers, and carefully taking out the chick embryo by using the tweezers;

(2) gently scraping skin of leg with scalpel, separating chick embryo leg, and placing in 1640 (primary myoblast culture medium) culture medium containing 20% fetal bovine serum;

(3) separating with forceps, discarding skin and bone, placing in a 1.5 μ L centrifuge tube, placing 3-4 leg muscles in one centrifuge tube, adding 500 μ L culture medium into each tube, and shearing leg muscle tissue with scissors until 1mL of gun head can be sucked up;

(4) transferring the minced muscle tissue into a 50mL centrifuge tube by using a pipette, vortexing and shaking for 1min, standing for 2min again, and waiting until the muscle tissue slowly sinks to the bottom. Transferring the supernatant, and filtering the supernatant to a 50mL centrifuge tube through a 200-mesh filter screen;

(5) adding 7mL of primary myoblast culture solution to resuspend the muscle tissue, performing vortex oscillation for 1min, and repeating the step for 3-4 times;

(6) centrifuging the filtrate collected by filtration at 1050rpm for 4min, and removing the supernatant;

(7) adding appropriate amount of culture medium to resuspend the obtained cells, transferring to a new culture dish, standing at 37 deg.C and 5% CO₂Culturing in a cell culture box. And obtaining the purified myoblasts by a differential adherence method twice.

3.2 immunofluorescence identification of Primary myoblasts

Desmin is a myoblast specific expression protein, and shows green fluorescence after being specifically combined with myoblasts. DAPI is a nucleic acid dye that can pass through the cell membrane of a living cell into the cell and bind to deoxyribonucleic acid in the nucleus, which appears blue under a fluorescent microscope. Myoblasts are identified by immunofluorescence staining, and most of the cells obtained by separation are myoblasts which can be used for subsequent experiments. The immunofluorescence of the chick embryo myoblast comprises the following steps:

(1) when the density of primary myoblasts in a 24-pore plate reaches 90%, washing the cells for 5min for 3 times by using 100 mu L of PBS per hole;

(2) discarding PBS, adding 250 μ L of fixing solution (4% cold paraformaldehyde) into each well, and standing at room temperature for 20 min;

(3) adding appropriate amount of PBS, washing with PBS every 5min at room temperature for 3 times;

(4) adding 1mL of 0.1% Triton X-100 membrane breaking solution into each hole, breaking the membrane at room temperature for 15min, discarding the membrane breaking solution, washing with PBS once every 5min at room temperature, and washing for 3 times;

(5) adding BSA blocking solution, 200 μ L/well, and blocking at room temperature for 30 min;

(6) discarding the confining liquid, washing without PBS, adding desmin and staying overnight at 4 ℃;

(7) the next day, the plate was taken out of the refrigerator at 4 ℃ and washed with PBS once every 5min for 3 times, then Fluorescein Isothiocyanate (FITC) diluted 1:50 was added to each well, incubated for 1h in the dark, washed with PBS once every 5min for 3 times;

(8) the DAPI staining solution is 200 mu L/hole for staining the nucleus for 5 min. Washing with PBS every 5min for 3 times after staining;

(9) a drop of glycerol was placed on the slide glass, and the slide glass was observed under a fluorescent inverted microscope and photographed as shown in FIGS. 5A and 5B.

3.3 cell plating

(1) When the density of primary cells in the culture dish reaches 100%, discarding the culture medium on a sterile operating platform, washing each dish for 2 times by using 1mLPBS (multi-layered phosphate buffer solution), removing serum and dead cells, and then discarding by using a pipette;

(2) digesting 1mL of pancreatin in each dish for 30s, observing under an inverted microscope, slightly blowing and beating to make cells fall off after the cells shrink and become round, stopping digestion of a culture medium, filtering by using a 200-mesh gauze (a screen is connected with a 50mL centrifuge tube below), and centrifuging at 1050rpm at room temperature for 5 min;

(3) the supernatant was discarded, cells were vortexed by adding fresh 1640 medium containing 20% fetal calf serum, and the cell suspension was uniformly pipetted into a 24-well plate with a volume of 500. mu.L per well. Put in 5% CO at 37 DEG C₂Culturing in an incubator.

3.4 transfection of cells

5 haplotype recombinant plasmids and a pcDNA3.1 empty vector plasmid are transfected into primary myoblastsEffect of the same haplotype on the expression of let-7b in myoblasts, procedure for cell transfection

The steps of the specifications of Reagents are carried out, and the specific steps are as follows:

(1) inoculating primary myoblasts into a 24-pore plate, and performing a transfection experiment when the cell density reaches about 75%, wherein each haplotype is transfected with 4 repeats;

(2) Opti-MEM 50. mu.L/well, 600 ng/well, p 30002. mu.L/well per recombinant plasmid;

(3) dilute with 50. mu.L/well Opti-ME

Add 0.75. mu.L per well

(4) Mixing the components (2) and (3) and incubating at room temperature for 5 min;

(5) adding 50 mu L of mixed solution into each hole of cells, slightly shaking the cell culture plate for several times, uniformly mixing, and putting the 24-hole plate back to the cell culture box at 37 ℃ for culture;

(6) after 6h of transfection, the culture medium is discarded, PBS is used for washing once, and fresh 1640 culture medium containing 20% serum is added;

(7) after 48h of incubation, the cells were removed and subjected to the next test.

3.5 RNA extraction

(1) Discarding the culture medium in the 24-well plate, adding 1mL of Trizol solution, blowing for several times, and standing for 5min at room temperature;

(2) centrifuging at 12000r/m at 4 ℃ for 15min, and transferring the supernatant to a new 1.5mL EP tube;

(3) adding 200 μ L chloroform, shaking vigorously and mixing for 15s, and standing at room temperature for 5 min;

(4) centrifuging at 12000r/m for 15min at 4 deg.C, and transferring supernatant into new EP tube;

(5) adding isopropanol with the volume equal to that of the supernatant fluid of about 500mL, and standing at-80 ℃ for 30 min;

(6) centrifuging at 12000r/m at 4 deg.C for 10min, discarding supernatant, adding 75% ethanol 1mL, washing (shaking moderately and mixing) and precipitating;

(7) centrifuging at 7500r/m at 4 deg.C for 5min, removing supernatant, retaining precipitate, and naturally drying at room temperature for 5 min;

(8) the dried precipitate was dissolved in an appropriate amount (3 to 50. mu.L) of DEPC-treated water to obtain an RNA solution.

3.6 Total RNA concentration determination

Total RNA integrity detection can be detected by normal agarose gel electrophoresis as follows:

(1) preparation of a 1% agarose gel: weighing 0.4g of agar powder, adding 40mL of 1 XTAE, heating in a microwave oven for 1min, cooling to about 60 ℃, adding 1.5 μ L of ED for dyeing, mixing, shaking uniformly, pouring into a gel box, removing bubbles, and inserting a proper comb. Cooling at room temperature for 30min, and removing the comb after the gel is solidified;

(2) sample application: sucking 3 mu L of total RNA and 2 mu L of 6 × Loading Buffer, blowing the total RNA and 2 mu L of the 6 × Loading Buffer by using a pipette gun, adding the total RNA and the 6 × Loading Buffer into a spot sample well, carrying out electrophoresis for 12min at 120V by using 2K DNA Marker as a contrast (replacing with a newly-prepared electrophoresis solution before the electrophoresis is started);

(3) and (3) photographing: after the gel electrophoresis is finished, observing and taking a picture by using a gel imaging system, wherein the standard total RNA has three electrophoresis bands of 5SrRNA, 18srRNA and 28srRNA, if the three bands are complete, the total RNA extraction effect is better, and the standard total RNA can be used for subsequent tests;

(4) and (3) detecting the purity of the total RNA: the total RNA concentration extracted was measured by UV spectrophotometer, and the measurement was repeated at least 3 times for each sample, and the average was taken as its final concentration. According to the average value, each RNA sample was diluted to 1. mu.g/. mu.L with DEPC water and stored in an ultra-low temperature freezer at-80 ℃ for further use.

3.7 Total RNA reverse transcription

(1) The reagents shown in Table 8 below were added to the RNase free PCR tube;

(2) heating the transcription system at 42 deg.C for 60 min;

(3) after heating at 72 ℃ for 10min, the reverse transcription product cDNA is stored at-20 ℃ for later use.

TABLE 8 Total RNA reverse transcription reaction System

3.8 qPCR

The reaction system for qPCR detection of miRNA content in the samples is shown in Table 9 below, and was performed using a Bio-Rad CFX96 real-time fluorescent quantitation PCR instrument. The reaction procedure is as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s, and 40 cycles. And analyzing the dissolution curve immediately after 40 cycles, wherein the detection temperature is 65-95 ℃, the heating rate is 0.5 ℃/time, and the constant temperature time is 5 s/time.

TABLE 9 let-7b qPCR reaction System

3.9 data analysis processing of qPCR

And (4) analyzing the relative expression quantity of the gene by adopting a Ct value comparison method. Δ Ct ═ Ct (target gene) -Ct (reference gene); Δ Ct (experimental group) Δ Ct (control group), and the relative expression level is 2- Δ Ct.

The expression level of let-7b mature body after 5 haplotypes and one pcDNA3.1 empty vector plasmid are over-expressed to chicken primary myoblasts for 48h is calculated according to the method of fluorescence quantitative data relative expression level being 2-delta-Ct, and the result is shown in figure 6, wherein the relative expression level of let-7b mature sequence after AGCC haplotype transfection is 2.362, AACC haplotype is 3.188, GATC haplotype is 3.649, GACG haplotype is 3.052, GACC haplotype is 2.977, and pcDNA3.1 empty vector is only 1.054.

The above embodiments are preferred embodiments of the present invention, and all similar processes and equivalent variations to those of the present invention should fall within the scope of the present invention.

SEQUENCE LISTING

<110> institute of Buddha science and technology

<120> method for constructing haplotype chicken myoblasts with high let-7b mature body expression

<130> 2018.10.16

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 24

<212> DNA

<213> Artificial Synthesis

<400> 1

ggaattcgta aatccaccta gcgt 24

<210> 2

<211> 25

<212> DNA

<213> Artificial Synthesis

<400> 2

gctctagatg cccaggaaat atgaa 25

Claims (1)

1. A method for constructing haplotype chicken myoblasts with high let-7b mature body expression quantity is characterized by comprising the following steps:

1) haplotype expression vector cloning

a. Designing a primer pair according to the sequencing result of a let-7b precursor region gene sequence, wherein a target fragment comprises g.270G > A, g.526G > A, g.678T > C and g.710G > C4 mutation sites, and simultaneously carrying out haplotype gene PCR amplification after adding an enzyme digestion site and a protective base at the 5' end of the designed primer pair to obtain a PCR product;

b. recovering and purifying the PCR product to obtain purified DNA solution containing the target segment for later use;

c. adopting pcDNA3.1 strain to carry out bacterial liquid propagation, extracting pcDNA3.1 circular plasmid and obtaining a purified DNA sample;

d. carrying out double enzyme digestion treatment on the DNA solution containing the target fragment obtained in the step b and the DNA sample obtained in the step c respectively by using endonuclease EcoR I and Xba I, and obtaining a target gene fragment and a pcDNA3.1(+) plasmid vector after purifying double enzyme digestion products;

e. d, connecting the target gene fragment obtained in the step d with pcDNA3.1(+) plasmid vectors by using T4 DNA Ligase, converting the connection products, and performing shake propagation to obtain 5 successfully-constructed haplotype recombinant plasmids;

2) culture of primary myoblasts in chickens

Separating myoblasts from the eggs of the chickens which are hatched for 11d, and purifying for later use;

3) cell transfection

Transfection reagents are adopted to transfect 5 haplotype recombinant plasmids to the primary myoblasts of the chicken respectively, and 5 haplotype myoblasts with high let-7b mature body expression quantity are obtained.

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