CN106434912B - Application of microRNA marker related to litter size of pig - Google Patents

Abstract

The invention belongs to the technical field of livestock molecular biology, and provides application of a microRNA marker related to pig litter size, wherein miR-429, ZEB1 and LH beta are used as marker genes, and the relative farrowing capacity of different sow individuals is judged by comparing the relative expression amounts of the marker genes miR-429, ZEB1 and LH beta in hypophysis, and the individual with high expression amount of the marker gene miR-429 is higher than the individual with low expression amount of the marker gene miR-429. The specific regulation mechanism is that miR-429 indirectly promotes the expression of LH beta gene by inhibiting the expression of ZEB1 gene in pituitary, thereby improving the litter size of sows. According to the invention, the farrowing capacity of the sow, particularly the farrowing capacity of about Kjeldahl and southern Anhui black pigs, can be rapidly obtained according to the microRNA marker expression condition related to the litter size of the pig, so that the error of external environmental factors in judging the farrowing capacity is avoided, the complexity in the breeding process is reduced, and the breeding time is greatly shortened through the detection on the molecular level.

Description

Application of microRNA marker related to litter size of pig

Technical Field

The invention belongs to the technical field of livestock molecular biology, and relates to application of a marker gene miR-429 in downstream gene regulation in the aspect of sow farrowing capacity judgment.

Background

Since the 50 s of the twentieth century, the breeding work of domestic and foreign pigs has undergone the transition from fat type to lean meat type with remarkable results, but the progress of genetic improvement is very slow for the reproductive traits with low heritability and the meat quality traits which are difficult to be measured in vivo. Farrowing performance is one of the important factors that determine pig reproductive capacity and economy of the modern pig industry. According to the measurement and calculation of Clutter and the like according to the current breeding scale of the British pigs, as long as the litter size of the pigs is increased by 1-1.5, the British pig industry in British can obtain additional profits of 7 hundred million British pounds each year; therefore, the number of weaned piglets per sow can be increased with less additional investment, and the economic return of the pig-raising producers is increased. Therefore, the litter size trait of the pig is paid more attention by breeders and producers, and has become the key point of the current pig genetic breeding research in China.

The reproductive process of pigs mainly depends on the regulation of hypothalamic-pituitary-gonadal axis, which is influenced by the interaction between genes and the environment. MicroRNA is a small non-coding RNA with the length of about 21-24 nt, and is widely involved in life activities of reproduction, development, virus defense, cell proliferation and apoptosis, fat metabolism and the like of eukaryote through post-transcriptional regulation of target genes. In recent years, studies on pig miRNAs have been increasing, but mainly focused on growth and development, disease occurrence and other related aspects of pigs. The studies on the regulation of porcine reproductive performance by miRNAs are relatively rare and it is not clear how miRNAs function in the hypothalamic-pituitary-gonadal axis.

Disclosure of Invention

The invention aims to use approximately Kyoxia and Anhui black pigs as test objects, understand the expression difference and the expression rule of miR-429 in reproductive axis tissues (pituitary, hypothalamus, ovary, uterus and oviduct) of sows with high and low litter sizes through a real-time fluorescence quantitative PCR technology, analyze the action mechanism of the miRNAs possibly playing a role in which tissues, and provide reference for the breeding and utilization of candidate miRNAs and excellent varieties of pigs.

1. The invention provides application of a marker gene miR-429 in regulating downstream genes in the aspect of judging farrowing capacity of sows, wherein the downstream genes are ZEB1 and LH beta; the downstream gene is regulated and controlled by miR-429 to inhibit the expression of a target gene ZEB1, a negative regulation relation is formed between ZEB1 and LH beta gene, and the expression of the LH beta gene is indirectly up-regulated by miR-429 through inhibiting the expression of the target gene ZEB 1; the marker gene miR-429 is positively correlated with the farrowing capacity of the sow, the target gene ZEB1 is negatively correlated with the farrowing capacity of the sow, and the target gene LH beta is positively correlated with the farrowing capacity of the sow; the farrowing capacity of the sow is judged by analyzing the expression level of the marker gene miR-429.

2. The application provided by the 1, wherein the farrowing capacity of the sow is judged by analyzing the expression levels of a marker gene miR-429 and a target gene ZEB 1.

3. The application provided by the 1 judges the farrowing capacity of the sow by analyzing the expression levels of the marker gene miR-429 and the target gene LH beta.

4. The application provided by the 1 judges the farrowing capacity of the sow by analyzing the expression levels of the marker gene miR-429, the target gene LH beta and the target gene ZEB 1.

5. The application provided by any one of the above 1-4, the application method specifically comprises the following steps: (1) tissue RNA extraction: extracting oviduct and pituitary RNA of different individual sows; (2) designing a primer: respectively designing a q-RT-PCR primer ssc-miR-429 of a candidate gene miR-429, a q-RT-PCR primer ZEB1 of ZEB1 and a q-RT-PCR primer LH beta of LH beta according to a target gene to be analyzed; (3) reverse transcription to synthesize cDNA: synthesizing cDNA (complementary deoxyribonucleic acid) from the total RNA obtained in the reverse transcription step (1) by using a reverse transcription kit; (4) determining the relative expression amounts of the candidate genes miR-429, ZEB1 and LH beta in the oviduct and pituitary of the sow by adopting a q-RT-PCR method; (5) according to the expression level of miR-429 in the oviduct and the pituitary, the expression level of ZEB1 and LH beta in the pituitary; judging the relative high and low farrowing capacity among different sow individuals;

wherein the sequence of the primer ssc-miR-429 is shown in SEQ ID NO. 1;

the upstream sequence of the primer pair LH beta is shown as SEQ ID NO.2, and the downstream sequence is shown as SEQ ID NO. 3;

the upstream sequence of the primer pair ZEB1 is shown as SEQ ID NO.4, and the downstream sequence is shown as SEQ ID NO. 5.

6. The application provided by the 5, wherein the step (2) further comprises the design of an internal reference gene primer, wherein the internal reference gene of miR-429 is U6, and the primer sequence is shown in SEQ ID NO. 6; the internal reference genes of LH beta and ZEB1 are beta-Actin, the sequence of the upstream primer is shown as SEQ ID NO.7, and the sequence of the downstream primer is shown as SEQ ID NO. 8.

7. The use as provided in 5 above, wherein, in the step (3), the reverse transcription kit Invitrogen code: a11193051 carries out transcription turning, a reverse transcription system is 20 mu L, in a miR-429 reverse transcription process system, 1 mu L of total RNA, 2 mu L of SuperScriptR enzyme mixture, 4 mu L of reaction mixture and 20 mu L of ultrapure water are added; the reaction conditions are as follows: inactivating reverse transcriptase at 37 deg.C for 60min, and maintaining at 95 deg.C for 5 sec;

in a reverse transcription process system of LH beta and ZEB1, 2.0 mu L of total RNA, 1 mu L of gDNA Eraser, 2.0 mu L of 5 XgDNA Eraser Buffer and 20 mu L of ultrapure water are added; the reaction conditions are as follows: reacting at 37 ℃ for 15min, and maintaining at 85 ℃ for 5sec to inactivate reverse transcriptase;

the q-RT-PCR method in the step (4) comprises the following reaction system: miR-429 as a 20-L reaction system, EXPRESS

GreenER^TMqPCR green fluorescent dye 10 μ L, primer ssc-miR-429 2 μ L, cDNA 2 μ L adding ultrapure water to 20 μ L; LH beta reaction System, iTaq^TMUniversal

Green Supermix Green fluorescent dye 7.5 μ L, primer pair LH β upstream and downstream sequences 0.5 μ L each, cdna1.5 μ L, ultrapure water added to 20 uL: the ZEB1 reaction system is the same as LH beta, and the primer pair is ZEB 1;

the reaction process is as follows: miR-429 is performed for 40 cycles of 95 ℃ for 20s, 95 ℃ for 5s and 60 ℃ for 20s, and fluorescence values are collected at 60 ℃ for 20 s; LH beta and ZEB1 are at 95 ℃ for 3min, 95 ℃ for 5s, 60 ℃ for 30s for 40 cycles, and fluorescence values are collected at 60 ℃ for 30 s; wherein the sow breeds are Anhui south black pigs and Yorkshire pigs.

8. The invention also provides a research method of a marker gene miR-429 on a downstream gene regulation mechanism, the method takes southern Anhui black pigs and Yorkshire pigs as objects, and a dual-luciferase experiment determines that ssc-miR-429 in the pigs is targeted to a 3' -UTR region of ZEB 1; the targeting relationship of transcription factors ZEB1 and LH beta in pigs is confirmed by a gel migration test.

9. The method of claim 8, wherein the vector used in the dual luciferase gene comprises a luciferase reporter vector: pGL3-Control, restriction enzyme site Xba I; miRNA expression vector: pmr-mcherry, the used restriction enzyme cutting sites are Bgl II and Xba I; the primers comprise PGL3-ZEB1 and ssc-miR-429, wherein the upstream sequence of the primer PGL3-ZEB1 is shown as SEQ ID NO.9, and the downstream sequence is shown as SEQ ID NO. 10; the upstream sequence of the primer ssc-miR-429 is shown in SEQ ID NO.11, and the downstream sequence is shown in SEQ ID NO. 12.

10. The research method of the above 8, wherein the DNA probes in the gel migration test are T1 and T2, wherein the upstream sequence of T1 is shown as SEQ ID NO.13 and the downstream sequence is shown as SEQ ID NO. 14; the upstream sequence of T2 is shown in SEQ ID NO.15, and the downstream sequence is shown in SEQ ID NO. 16.

The concept of comparison provided by the application is comparison among individuals, and the farrowing capacity among different individuals can be determined according to the expression amounts of the marker gene miR-429, the target gene ZEB1 and LH beta among different individuals. "high" and "low" are obtained by comparing data between individuals and are a relative concept.

The invention has the following advantages:

1. the application obtains the targeting relationship among miR-429, ZEB1 and LH beta in the pig pituitary, fills the gap of the prior art, obtains the relationship between the farrowing capacity of the sow and the expression difference of the candidate genes, and provides reference for breed breeding and new strain establishment in the future.

2. According to the invention, the difference of miR-429 expression amounts in the pituitary and the oviduct in the sows, particularly southern Anhui black pigs and Yorkshire pigs, can be used for rapidly judging the litter size between different sow individuals, and the difference of ZEB1 and LH beta expression amounts in the pituitary can be combined to more accurately and rapidly compare the litter size between different sow individuals, so that the error of the judgment of the litter size by external environmental factors can be avoided, the complexity in the breeding process can be reduced, and the breeding time can be greatly shortened by the detection on the molecular level.

Drawings

FIG. 1 Total RNA assay results.

FIG. 2 expression pattern of miR-429 in ovary, uterus, oviduct, hypothalamus, pituitary of approximately Kyoxia and Anhui black pigs. Wherein ". star" shows a significant difference (P < 0.01), ". star" shows a significant difference (P < 0.05).

FIG. 3 differential analysis of miR-429 expression in ovary, uterus, oviduct, hypothalamus and pituitary of about Kyoxia high and low farrowing sows. Wherein ". star" shows a significant difference (P < 0.01), ". star" shows a significant difference (P < 0.05).

FIG. 4 is an analysis of expression difference of miR-429 in ovaries, uteri, oviducts, hypothalamus and pituitary of high-and-low-yield sows in southern Anhui. Wherein ". star" shows a significant difference (P < 0.01), ". star" shows a significant difference (P < 0.05).

FIG. 5 pGL3-Control vector map.

FIG. 6 pmr-mcherry vector map.

FIG. 7 expression difference of ZEB1 in tissues of ovary, uterus, oviduct, hypothalamus, pituitary and the like of approximately Kyoto high litter group and low litter group. Wherein ". star" shows a significant difference (P < 0.01), ". star" shows a significant difference (P < 0.05).

FIG. 8 shows the difference in LH β expression in the tissues of ovary, uterus, oviduct, hypothalamus, pituitary and the like in approximately Kyowa litter group and litter group. Wherein ". star" shows a significant difference (P < 0.01), ". star" shows a significant difference (P < 0.05).

FIG. 9 expression and differential analysis of ZEB1 in ovary, uterus, oviduct, hypothalamus, pituitary and other tissues of southern Anhui black pig. Wherein ". star" shows a significant difference (P < 0.01), ". star" shows a significant difference (P < 0.05).

FIG. 10 expression and differential analysis of LH β in the ovary, uterus, oviduct, hypothalamus, pituitary and other tissues of southern Anhui black pig. Wherein, the difference is extremely significant (P < 0.01) ", the difference is significant (P < 0.05)")

FIG. 11 ssc-miR-429 and ZEB1 determined sequences were compared with the designed sequences. Wherein, the graph A shows the sequence comparison of ssc-miR-429 and the sequence cloned to the miRNA expression vector pmr-mcherry; panel B shows a sequence comparison of ZEB1 and the cloned miRNA expression vector bifluorescent detection vector pGL 3-Control.

FIG. 12 transfection effects of different concentrations of Lipofectamine TM 2000. Wherein, Panel A shows the transfection effect at a concentration of 1 ul; panel B shows the effect of transfection at a concentration of 2 ul; panel C shows the effect of transfection at a concentration of 5 ul.

FIG. 13 fluorescence ratio of empty vector and vector with ssc-miR-429. Wherein ". star" is very significant in the difference (P < 0.01).

FIG. 14 is a standard curve prepared from absorbance data of standard substance with different concentrations measured by microplate reader A562

FIG. 15 EMSA results of interaction of DNA probes T1, T2 with white pig uterine nucleoprotein. Wherein 1 is a null probe, 2 is a competition, 3 is T1, 4 is a null probe, 5 is a competition T2, and 6 is T2.

Detailed Description

The present invention will be specifically described with reference to the following examples.

Example 1:

1.1 test subjects

The used joker summer and southern Anhui black pigs for this experiment were taken from the pig farm of the university of agriculture in Anhui. Carrying out normal distribution analysis by counting the number born of the sow, selecting 3 healthy sows which have the number born distributed at two tails and consistent body condition and age in the intermission period, slaughtering and taking tissues such as ovary, pituitary, hypothalamus, oviduct, uterus and the like, and storing by liquid nitrogen for extracting total RNA. Sample information is shown in table 1.

TABLE 1 sample information

Note: the average litter size in the tables is the mean. + -. standard error

1.2 Main instruments and test reagents

1.2.1 Main Instrument

See table 2.

TABLE 2 Main instruments

1.2.2 Primary reagents

See Table 3

TABLE 3 major reagents

Wherein the reagents such as absolute ethyl alcohol, isopropanol, chloroform, DEPC treated water, agarose, 10 xTBE and the like are imported or domestic split charging reagents and are analytically pure.

1.3 Experimental methods

1.3.1 miR-429 primer design and Synthesis

Designing an upstream specific primer by a tailing method according to a mature sequence of miR-429 of a pig in a latest miRase database, wherein a downstream general primer is provided by an Invitrogen kit; through the experimental result of a fluorescent quantitative standard curve, U6 is selected as an internal reference gene of miRNAs to carry out RT-PCR, and experimental primers are synthesized by Shanghai Production gene company.

TABLE 4 primer sequences for genes and miRNAs

Note: f is an upstream primer, R is a downstream primer

1.3.2 Total RNA extraction and reverse transcription

1.3.2.1 Total RNA extraction

The total RNA extraction in this experiment was performed by TRIzol (Invitrogen) extraction. The operation steps are as follows:

after the sample is rapidly ground by liquid nitrogen, a proper amount of tissue is added into 1ml of TRIzol, the mixture is shaken and kept stand at room temperature to be fully cracked, the mixture is centrifuged at 13000rpm for 5min, and then the supernatant in the tube is transferred to a new 2.0ml of EP tube (if the sample is fat-rich tissue, the grease on the upper layer needs to be removed).

Adding chloroform, covering the tube cover tightly, shaking and mixing uniformly on a vortex oscillator, and standing at room temperature to naturally split phases.

Centrifugation at 13000rpm at 4 ℃ will result in a mixture that will separate into three layers: the lower red layer is phenol-chloroform organic phase, the middle layer and colorless upper aqueous phase, and RNA is mainly concentrated in the aqueous phase.

The supernatant was transferred to a new 2.0ml EP tube (care was taken not to suck the middle and lower layers), chloroform was added, the mixture was shaken and mixed on a vortex shaker for 15 seconds, allowed to stand at room temperature to allow natural phase separation, and centrifuged at 13000rpm at 4 ℃.

Transferring the upper layer water phase into a new 1.5ml EP tube, marking the project name, sample name and date on a centrifuge tube, adding isopropanol with the same volume, mixing uniformly, and standing at-80 ℃ or dry ice for a period of time.

Centrifugation was carried out at 13000rpm at 4 ℃ to discard the supernatant.

Adding 1ml of 75% ethanol into the RNA precipitate, reversing and uniformly mixing for 5s, centrifuging at 4 ℃ and 13000rpm for 5min, and removing the supernatant; and 7, repeating the step.

Centrifuge at 13000rpm for 1min at 4 ℃ and carefully discard the excess liquid with a pipette tip.

Drying the RNA precipitate in an ultraclean workbench for about 1-2min (the RNA is not easy to dry, otherwise the RNA is difficult to dissolve), dissolving the RNA precipitate with a proper amount of RNase-Free ddH2O, gently blowing the precipitate with a pipette if necessary, and storing at-80 ℃ after the RNA precipitate is completely dissolved.

1.3.2.2RNA quality testing

1) And (3) detecting the concentration and purity of the RNA: taking 1 mu L of RNA solution to be detected, and determining the concentration and purity of the RNA on an ultramicro ultraviolet spectrophotometer. RNA purity OD260/OD280, purity between 1.8-2.0 as qualified RNA sample, can be reverse transcription. If the purity value is lower than 1.8, protein pollution exists; if the purity value is higher than 2.0, there is contamination with other nucleic acids.

2) Detecting RNA quality by formaldehyde denatured gel: 2g agarose was weighed, 20mL 10 XTBE, 144mL DEPC water was added, the mixture was heated to complete dissolution in a microwave oven, 5. mu.L 10mg/mL EB solution was added after cooling to about 60 ℃, 36mL formaldehyde was added in a fume hood, and after standing for a period of time, gelation was carried out. Taking 4 mu L of RNA sample, adding 1 mu L of 5 XRNA Loading buffer, heating in a 70 ℃ water bath kettle for 10min, placing on ice for quenching, placing the mixed sample on formaldehyde denaturing gel, carrying out electrophoresis detection at 3-5V/cm, and observing an electrophoresis strip.

1.3.2.3 reverse transcription and quantitation of miRNA

A reverse transcription reaction solution was prepared using a reverse transcription kit (Invitrogen code: A11193051) by the following composition, as shown in Table 6.

TABLE 6 miRNA reverse transcription reaction protocols

The reaction conditions for the reverse transcription were as follows: the reaction was maintained at 37 ℃ for 60min and 95 ℃ for 5sec (reverse transcriptase inactivation reaction). After the sample cDNA obtained after reverse transcription was used for PCR detection, RNase Free water was added to 40. mu.L. The reverse transcription product was stored in a freezer at-20 ℃.

The expression quantity of the pig miR-429 and internal reference gene U6 genes is detected by a real-time fluorescent quantitative PCR method, and the reaction is carried out on a Bio-rad CFX96 qPCR fluorescent quantitative PCR system. The reaction system was 20. mu.L, and the specific procedures are shown in Table 7:

TABLE 7 RT-PCR reaction procedure

Each sample was repeated 3 times. After the reaction cycle was completed, the product purity of RT-PCR was checked by the dissolution profile.

1.3.2.4 Gene expression data analysis

Analysis of expression level of Gene: the statistical analysis of the validity data was carried out by the 2- Δ Δ Ct method. The relative expression quantity of the target gene is 2-delta CT, the delta Ct represents an internal reference gene, wherein the miRNA takes U6 as the internal reference gene, the average value of the Ct difference values of the target gene and the internal reference gene of a control group is taken as a reference value, x represents any sample, and the formula is as follows:

ΔΔCt＝(Ct.Target gene-Ct.Reference gene)x-(Ct.Target gene-Ct.Reference gene)control

the expression quantity of the target gene of each sample is calculated by the formula, after the internal reference gene is corrected, the data is expressed by mean value plus or minus standard error (M plus or minus SE), the results of experimental data are statistically analyzed and plotted by GraphPad Prism 6 software, and the significance of the mean value difference is analyzed and compared by a t-test method. P < 0.01 indicates a significant level of difference, and P < 0.05 indicates a significant level of difference.

1.4 results and analysis

1.4.1 extraction and detection of Total RNA

As can be seen from the figure, the extracted total RNA is detected by 1% agarose gel electrophoresis, and three clear bands are seen, namely 28S, 18S and 5S rRNA from top to bottom. The ratio of OD260/OD280 is 1.9-2.1, which indicates that the extracted total RNA has good quality and no pollution of protein and other impurities, and can be used for carrying out subsequent reverse transcription tests.

1.4.2 expression differences of miR-429 in reproductive axis tissues of two different varieties of sows

Quantitative experiments show that the high expression tissues of the miR-429 are different. miR-429 is highly expressed only in the fallopian tubes and pituitary. In the same tissue, miR-429 expression in approximately Kwangsu pituitary is significantly lower than that of southern Anhui black pig (P < 0.01), and expression in approximately Kwangsu oviduct is significantly higher than that of southern Anhui black pig (P < 0.01).

1.4.3 expression rule of miR-429 in reproductive axis tissues of sows with different litter sizes of the same variety

In approximately g, the expression of miR-429 in both the fallopian tube and pituitary in the high-yielding group was higher than that in the low-yielding group (P < 0.01 or P < 0.05). In southern Anhui black pigs, the expression of miR-429 in both pituitary and oviduct of the high-yielding group was higher than that of the low-yielding group (P < 0.01).

1.5 discussion

1.5.1 Effect of miR-429 on litter size in sows

miR-429 belongs to miR-200 family, and the research thereof mostly focuses on the research of human cancers. Research in pigs, particularly in the breeding of pigs, is very scarce. According to quantitative data of miR-429 in tissues of a reproductive axis in the experiment, miR-429 is almost only expressed in two tissues of an oviduct and a pituitary, and the expression of a high-yield group is higher than that of a low-yield group in the two tissues. Suggesting that miR-429 probably exerts a regulatory effect on litter traits in the pituitary and hypothalamus.

The oviduct has extremely complex and fine physiological functions and plays an important role in egg picking, sperm capacitation, ovum fertilization, fertilized egg delivery and survival and development of early embryos, so the condition of the oviduct has important significance in the smooth progress of reproductive behaviors. We can conclude that miR-429 might have an up-regulating effect on the litter size of sows in the pituitary and oviducts of sows.

1.6 summary knot

The experiment discusses the expression rule of miR-429 in the reproductive axis tissue of the sow and the expression difference of the miR-429 in high-farrowing sow groups and low-farrowing sow groups. By analyzing quantitative data of miR-429 in reproductive axis tissues of sows with different litter sizes of two breeds, the miR-429 can possibly generate an up-regulation effect on the farrowing traits of the sows in oviducts and hypophysis of the sows.

Example 2

2.1 test materials

The experimental samples used in the real-time fluorescence quantification experiment were the same as in example 1.

In addition, 1 healthy koxia sow is selected from a pig farm in the experiment base of Anhui agricultural university, tissues such as uterus and heart are slaughtered and stored in liquid nitrogen. Instructions for use:

since the quantitative test of example 1 showed that ZEB1 has a high level of expression in the uterus, the uterus cells of the Yorkshire sow were selected for extracting nucleoprotein for EMSA test.

DNA is extracted from heart tissue of about Kyara sow and used for preparing carrier required by dual-luciferase experiment

2.2 preparation of common instruments and reagents

Common instruments are shown in Table 8.

TABLE 8 common instruments

The usual reagents are shown in Table 9.

TABLE 9 common reagents

The following reagents were also prepared:

(1) 1.0% agarose electrophoresis gel: 0.5g agarose is weighed and added into 50mL IXTBE electrophoresis buffer solution, the mixture is fully heated and dissolved in a microwave oven, 2-3ul Gelstain is added after the mixture is cooled to 60 ℃, and the mixture is fully mixed.

(2) Preparation of 6% non-denatured polyacrylamide gel: in a 15ml test tube, ddH2O7.15ml, 30% polyacrylamide solution 2ml, 5 XTBE 1ml, glycerol 175ul, 10% APS 75ul, TEMED 8ul were placed in this order and mixed well.

1.3 Experimental methods

1.3.1 real-time fluorescent quantitation primer design and Synthesis of ZEB1 and LH β

Designing upstream and downstream primers of a target gene by using Primer Premier 5.0 software according to mRNA sequences (sequence numbers are XM-003482813.2 and NM-214080.1) of ZEB1 and LH beta of pigs in a GenBank database; and through the experimental result of a fluorescent quantitative standard curve, selecting beta-Actin (with the sequence number of XM-003124280.2) as the internal reference genes of the mRNAs to carry out RT-PCR, and synthesizing the primers of each gene by Shanghai. The primers for the upstream and downstream of each target gene are shown in Table 10.

TABLE 10 primers used in the assay

Note: f is an upstream primer, R is a downstream primer

2.3.2 Dual-luciferase vector and target fragment primer design

The carrier used in the dual-luciferase experiment is responsible for construction by the hybrid fertilizer Walang Biotech company:

(1) luciferase reporter vector: pGL3-Control, restriction site Xba I used. pGL3-Control vector is shown in FIG. 5:

(2) miRNA expression vector: the cleavage site used was Bgl II & Xba I. The pmr-mcherry vector is as in FIG. 6:

combining the prediction results of the miRNA and the target gene ZEB 13' UTR and the information of the used vector and enzyme cutting site to design primers, wherein the primers are synthesized by Shanghai worker, and the specific information of the primers is shown in Table 11:

TABLE 11 primers for Dual luciferase assay

Note: f is an upstream primer, R is a downstream primer

2.3.3 DNA Probe design for EMSA

Combining the combination condition of the transcription factor ZEB1 and the LH beta promoter region of the target gene, selecting two predicted regions to prepare probes T1 and T2, wherein the DNA probe used in the experiment is synthesized by Nanjing Dingding biology, and the sequence information of the probe is shown in Table 12.

TABLE 12 Probe primers for EMSA

Note: f is a forward primer, R is a reverse primer

2.3.4 reverse transcription and quantitation of ZEB1 and LH β

Total mRNA used in this reverse transcription experiment was extracted from run one and the reverse transcription reaction was performed according to the PrimeScriptTM RT reagent kit by TaKaRa with gDNA Eraser (Peffect Real Time) reagent instructions:

the first step is to remove the DNA from the total RNA. mu.L (250 ng/. mu.L) total RNA, 2. mu.L 5 XgDNA Eraser Buffer, 1. mu.L gDNA Eraser were added to a 0.2ml RNase-Free PCR tube, 5. mu.L RNase Free dH2O was added to the tube to a volume of 10. mu.L, centrifuged and placed in a PCR apparatus, pre-denatured at 42 ℃ for 2min, and stored at 4 ℃. The reaction system for removing genomic DNA is shown in Table 13.

TABLE 13 removal of genomic DNA reaction

And secondly, carrying out reverse transcription reaction on the treated product. To the first step reaction was added 4. mu.L of 5 XPrimeScript Buffer 2(for Real Time), 1. mu.L of PrimeScript RT Enzyme Mix I, 1. mu.L of RT Primer Mix, 4. mu.L of RNase Free dH2O in total volume of 20. mu.L, mixed and centrifuged briefly, and the reaction tube was reacted in a PCR apparatus at 37 ℃ for 15min and at 85 ℃/5sec to inactivate the reverse transcriptase. The reverse transcription product (RT product) was stored at-20 ℃ for real-time quantitative PCR after packaging. mRNA reverse transcription system is shown in Table 14.

TABLE 14 mRNA reverse transcription System

The expression levels of ZEB1, LH beta and the internal reference gene beta-Actin of the pig are detected by a real-time fluorescent quantitative PCR method, and the reaction is carried out on a Bio-rad CFX96 qPCR fluorescent quantitative PCR system. The reaction was 20. mu.L, and each sample was repeated 3 times. After the completion of the reaction cycle, the gene quantitative data was processed in the same manner as in example 1.

The specific steps are shown in Table 15.

Table 15 RT-PCR reaction protocol for mRNA

2.3.5 Dual luciferase reporter Gene experiments

2.3.5.1 extraction and sequencing identification of recombinant plasmid

The experiment was performed according to the instructions of the Kit for easy pure Plasmid MiniPrep Kit of Beijing holotype gold organisms:

the overnight culture was removed, centrifuged at 10,000Xg for 1min, and the supernatant removed (as far as possible). If the bacterial liquid brightness is too large, the bacterial liquid can be collected centrifugally for many times.

Adding colorless solution RB (containing RNase A), and shaking to suspend the thallus, wherein small bacterium blocks are not remained.

Adding blue solution LB, turning and mixing 4-6 times to crack thallus completely to form blue bright solution, changing color from semi-bright to bright blue, indicating complete cracking (not more than 5 min).

Add yellow NB solution, mix gently 5-6 times (color change from blue to yellow indicating uniform mixing and complete neutralization) until a compact yellow clump is formed, and stand 2 minutes at room temperature.

Centrifuge at 12,000Xg for 5 minutes, carefully aspirate the supernatant and add to the spin column. Centrifuge at 12,000Xg for 1 minute and discard the effluent. (if the volume of the supernatant is more than 800ul, it can be added to the column in several portions and centrifuged as above to discard the effluent).

250ul of TB solution was added, and the mixture was allowed to stand at room temperature for 10 minutes, centrifuged at 12,000Xg for 1 minute, and the effluent was discarded.

650ul of WB solution was added, and the mixture was centrifuged at 12,000Xg for 1min, and the effluent was discarded.

Centrifugation at 12,000Xg for 1-2 minutes thoroughly removed residual WB.

The column was placed in a clean centrifuge tube and 30-50ul EB or deionized water (pH > 7.0) was added to the center of the column and allowed to stand at room temperature for 1 minute. (EB or deionized water is preheated in water bath at the temperature of 60-70 ℃, and the using effect is better).

10,000Xg centrifugation for 1min, elution of DNA at-20 ℃ storage for sequencing.

2.3.5.2 optimization of Co-transfection conditions

The efficiency of cell cotransfection is influenced by the quantity of liposome, the ratio of liposome to carrier, experimental cells and other factors. This experiment was most suitable for transfection experiments at a cell density of 70% based on previous experimental experience using six well plates with a recommended addition of 2ug of plasmid per well (1ug pmr-mcherry or pmr-mcherry-ssc-mir-429+1ug pGL3-Control-ZEB 1). To find the optimal amount of liposomes added per well, we set 3 different doses of Lipofectamine TM2000 at 1ul, 2ul, 5ul according to the literature and then observe the transfection efficiency.

Before transfection, the cell culture solution was changed to DMEM medium without double antibody, and 293T cells were transfected with plasmids at a cell density of 70%. 125ul of OPTI-MEM was added to 3 EP tubes, 1ul, 2ul and 5ul of Lipofectamine TM2000 were added, mixed by shaking and left to stand for 5 min. 2ug of plasmid (pmr-mcherry-ssc-mir-429+1ug of pGL3-Control-ZEB1) was added to each tube, mixed by shaking, and allowed to stand for 20 min. The mixture after standing is added into a six-well plate, fresh culture solution is replaced after 6 hours, fluorescence is observed for 24 hours, and transfection efficiency is confirmed. Wherein the pmr-mcherry-ssc-mir-429 plasmid carries a mcherry fluorescent dye, and the red light can be used for directly observing the transfection effect under an inverted microscope.

2.3.5.3 cotransfection and dual-luciferase reporter gene activity detection

After determining the optimal addition of Lipofectamine (TM) 2000, two sets of co-transfections were performed in 6-well plates: 1ug pGL3-Control-ZEB1+1ug pmr-mcherry-ssc-mir-429or pmr-mcherry. To ensure the accuracy and reliability of the experiment, we performed 5 biological replicates per group. And (3) replacing fresh culture solution after 6 hours of transfection, and detecting the fluorescence values of luciferase and renilla by using a dual-luciferase reporter gene detection system after 24 hours. Where Renilla fluorescence values were used as an internal control, normalization was performed to eliminate differences in cell number and transfection efficiency.

The method for detecting the fluorescence activity of the Dual luciferase reporter assay system comprises the following steps:

cell lysis: after sucking up the cell culture solution, directly adding 100ml of fully and uniformly mixed reporter gene cell lysate into each hole of 293T cells cultured in a 6-hole plate, and fully lysing the cells;

dissolving the firefly luciferase detection reagent and the renilla luciferase detection buffer solution, and reaching the room temperature. Renilla luciferase assay substrate (100X) was placed in an ice bath or ice box for use.

According to the amount of 100ul required by each sample, a proper amount of renilla luciferase detection buffer solution is taken, and renilla luciferase detection substrate (100X) is added according to the ratio of 1: 100 to prepare the renilla luciferase detection working solution.

The fluorescence detector was turned on according to the instructions, and the measurement time interval was set to 2s and the measurement time was set to 10s.

For each sample, 100ul of firefly luciferase assay reagent was added, and rlu (relative light unit) was measured after mixing well by gun blow. Reporter cell lysates were used as blank controls.

After the above steps are completed, 100ul of renilla luciferase detection working solution is added, and RLU is measured after the solution is evenly beaten by a pipette

In the case of renilla luciferase as an internal control, RLU values measured with firefly luciferase were divided by RLU values measured with renilla luciferase. The degree of activation of the reporter gene of interest is compared based on the obtained ratio.

2.3.6 EMSA experiment

2.3.6.1 extraction of nucleoprotein

The experiment was carried out according to the instructions of the NE-PER Nuclear and cytological Extraction Reagents kit from Thermo Scientific:

(1) flushing a large white pig uterine tissue sample by using precooled PBS; transferring to a1.5 ml centrifuge tube, centrifuging for 3min at 500 Xg; carefully discarding the supernatant for later use;

(2) adding 100ul of precooled CER I (containing Protease Inhibitor); violent votex 15s, ice bath for 10 min;

(3) adding precooled CER II 5.5 ul; violent votex 5s, ice bath for 1 min;

(4) vigorous votex 5s, 16000 Xg, centrifuge for 10 min;

(5) immediately sucking the supernatant into a precooled centrifugal tube to obtain the tissue plasma protein obtained by extraction;

(6) adding 40ul of precooled NER (containing Protease Inhibitor);

(7) violent votex 15s, placed on ice, violent votex 15s every 10min for 40 min;

(8)16000 Xg, centrifuging for 10 min; immediately sucking the supernatant into a precooled centrifugal tube to obtain the tissue nucleoprotein obtained by extraction.

2.3.6.2 protein concentration detection using BCA:

the experiment was performed according to the BCA protein quantification kit instructions of Peking Edley:

according to the quantity of samples, preparing a proper amount of BCA working Solution according to 50 times of volume Solution A and 1 time of volume Solution B (50: 1), and fully and uniformly mixing.

And after the protein B standard substance is completely dissolved, adding the diluted standard substance into a 96-well plate according to the ratio of 0, 1, 2, 4, 8, 12, 16 and 20ul respectively, and adding the diluent to make up to 20 ul.

And C, adding 200ul of BCA working solution into each hole, slightly blowing, uniformly mixing, and standing at 37 ℃ for 45 min.

After cooling to room temperature, absorbance of A562 was measured by a microplate reader. And a standard curve is made according to the measured data to calculate the protein concentration in the sample.

2.3.6.3 EMSA experiments on ZEB1 and LHB

(1) Preparation of 6% non-denatured polyacrylamide gel: in a 15ml test tube, ddH2O 7.15.15 ml, 30% polyacrylamide solution 2ml, 5 XTBE 1ml, glycerol 175ul, 10% APS 75ul, TEMED 8ul are put in sequence;

(2) pre-electrophoresis at 4 deg.c and 100V voltage for 60 min;

(3) preparing a combined reaction solution according to the requirements of an EMSA kit (cargo number: 20148) of PIERCE company, and standing at room temperature for 10 min;

(4) adding biotin-labeled probe, and standing at room temperature for 45 min;

(5) adding 5ul of Loading Buffer into each binding reaction, mixing uniformly, and Loading;

(6) electrophoresis at 4 ℃ and 100V until the length of the gel is 2/3 after bromfenglan electrophoresis;

(7) film transfer: soaking nylon membrane with proper size in 0.5 × TBE for 15min, sequentially placing sponge, filter paper, gel, nylon membrane, filter paper and sponge according to sandwich method, fixing sieve plate, carefully removing all bubbles in sandwich, inserting gel into electrophoresis tank facing cathode, pouring 0.5 × TBE, and transferring under constant current of 300mA for 30 min; ultraviolet crosslinking;

(8) detection of biotin-labeled DNA by chemiluminescence: reference PIERCE

The Chemilmescent EMSA Kit states the operation;

(9) gently preheating a blocking Buffer (blocking Buffer) and a 4 Xrinsing Buffer (Wash Buffer) in a water bath at 50 ℃ until the particles are completely dissolved;

(10) placing the membrane in a clean plastic plate, adding 20ml of sealing buffer solution, and placing on a shaking table to gently shake for 15 min;

(11) sucking 66.7ul of horseradish streptavidin peroxidase substrate into 20ml of sealing buffer solution, and uniformly mixing for later use;

(12) pouring the blocking buffer solution, adding the solution prepared in the step 10, and placing on a shaking table to gently shake for 15 min;

(13) taking 40ml of 4 rinsing buffer solution to 120ml of ddH2O to obtain 1 rinsing buffer solution; transferring the membrane to a new plastic plate, adding 20ml1 rinsing buffer solution, rinsing for 5min, rinsing for 4 times;

(14) transferring the membrane to another new plastic plate, adding 30ml substrate balance buffer solution, and balancing for 5 min;

(15) preparing a Substrate Working Solution (Substrate Working Solution): adding 6ml Luminol/Enhancer Solution into 6ml Stable Peroxide Solution, mixing, and keeping away from light for use;

(16) taking out the membrane from the substrate balance buffer solution, placing the membrane into a new clean plate with the right side facing upwards, pouring the substrate working solution into the surface of the membrane, and placing the membrane for 5min in a dark place;

(17) taking the membrane out of the substrate working solution, and sealing the membrane by using a plastic packaging membrane to avoid bubbles and wrinkles;

(18) the film was exposed to X-ray film in a dark room, developed, fixed, developed, and the results were recorded by exposure.

2.4 results

2.4.1 expression rule of ZEB1 and LH beta genes in reproductive axis tissues of sows of the same breed with different litter sizes

As shown in FIGS. 7-8, the expression of pituitary ZEB1 in the high yield group was significantly lower than that in the low yield group (P < 0.01) in about g. The expression of pituitary LH beta in the high-yield group is remarkably higher than that in the low-yield group (P is less than 0.01).

As shown in FIGS. 9-10, in southern Anhui black pigs, the expression of ZEB1 was lower in the pituitary gland of the high-yielding group than in the low-yielding group (P < 0.01). LH beta is expressed mainly in pituitary gland with tissue specificity, and the high-yielding group is higher than the low-yielding group (P < 0.01).

2.4.2 Dual luciferase assay

2.4.2.1 sequencing results and analysis of recombinant plasmids

According to the sequence comparison of the fragment sequence obtained by designing a primer and a sequencing result, ssc-miR-429 and ZEB 13' UTR are successfully cloned into a miRNA expression vector pmr-mcherry and a double-fluorescein detection vector pGL3-Control respectively, the determined sequences are completely consistent with the design as shown in figure 11, and the sequence can be used in a subsequent double-fluorescence detection experiment.

2.4.2.2 optimization of transfection conditions

In the experiment, 3 experimental groups A, B and C with different addition amounts are established according to the optimal addition amount of Lipofectamine TM 2000. Since the transfected pmr-mcherry-ssc-mir-429 is a protein with mcherry fluorescence, the fluorescence luminescence read-out level can be observed under an inverted microscope to evaluate the transfection efficiency. The concrete results are shown in FIG. 12

The results showed that the transfection efficiency was highest at 5ul/well of Lipofectamine TM 2000. Thus in the following co-transfection experiment, transfection was performed using Lipofectamine (TM) 2000 at 5 ul/well.

2.4.2.3 detection of dual-luciferase reporter gene activity

The experiment was performed according to the protocol of the Promega Dual Luciferase reporter assay 1910 Dual fluorescent reporter System with Fluoroskan assay FL fluorescent chemiluminescent microplate reader to obtain fluorescence values, all Luciferase fluorescence values in the raw data were then normalized with Renilla and plotted with GraphPad Prism 6 FIG. 13

As can be seen from FIG. 13, the fluorescence ratio between the empty vector experimental group and the carrier with ssc-miR-429 experimental group shows that the fluorescence value of the empty vector experimental group is very significantly higher than that of the carrier with ssc-miR-429 experimental group (P < 0.01). Indicating that ssc-miR-429 targets the 3' -UTR region of ZEB 1.

24.3 EMSA experiment

2.4.3 concentration of BCA assay protein

According to the measurement method provided by the BCA kit: measuring absorbance of standard substance with different concentrations in A562 with microplate reader, and making standard curve according to data

TABLE 16 absorbance of standards of different concentrations determined by BCA method

According to the standard curve and the absorbance of the sample A562, the concentration of the protein extracted from the uterus of the white pig is calculated to be 16.46mg/ml, which meets the requirements of the next EMSA experiment.

2.4.4 EMSA test identification results

The EMSA results showed that bands in which the DNA probe was bound to the target protein and migrated appeared in the gel wells of FIG. 15, T1 and T2, indicating that transcription factor elements capable of specifically binding to the DNA probe did exist in the nuclear protein of the sample. However, the extracted nucleoprotein contained endogenous biotin, so that both competitive T1 and competitive T2 showed bands.

From the quantitative data of miR-429, ZEB1 and LH beta in the test results, miR-429 has the tendency of down-regulating ZEB1 in the pituitary and ZEB1 also has the tendency of down-regulating LH beta, so that miR-429 possibly has a regulation mechanism for up-regulating the fertility candidate gene-LH beta by down-regulating the target gene ZEB1 in the pituitary of pigs.

The miRNA can perform post-transcriptional regulation through a 3' UTR region of a target gene, thereby playing a role in regulating the protein expression level of the target gene. According to the present example, the fluorescence value of the empty vector group was higher than that of the vector group with ssc-miR-429 (P < 0.01), and we can confirm that there is a targeting relationship between miR-429 and ZEB1 in pigs, and that the targeting relationship can be up-regulated. As a result of EMSA binding in the test, we found that a band in which the probe binds to the target protein and migrates appears only in the electrophoresis lanes of the specific DNA probes T1 and T2, indicating that transcription factor elements capable of specifically binding to the DNA probes do exist in the nuclear protein of the sample. This makes more clear the targeting concerns of the transcription factors ZEB1 and LH β. In conclusion, the existing experimental results are utilized by the inventor to preliminarily verify that miR-429 can regulate the LH beta up by down-regulating ZEB1 in the pituitary.

The above example firstly analyzes the expression rule of miR-429 in reproductive tissues of high-low yield about Kyoxia and southern Anhui black pigs, and deduces that miR-429 may influence the farrowing performance of pigs. And then, verifying the molecular mechanism of miR-429 for regulating the piglet farrowing characters through a plurality of verification experiments such as real-time fluorescence quantification, dual-luciferase reporter genes, electrophoretic mobility and the like. To obtain:

(1) miR-429 participates in pig reproduction regulation;

(2) miR-429 can indirectly regulate the expression of LH beta gene by inhibiting the expression of a target gene ZEB1, thereby regulating the farrowing performance of pigs.

It will be understood that the above-described embodiments are merely illustrative of the principles of the invention, which is not limited thereto, and that various modifications and changes can be made by those skilled in the art without departing from the spirit of the invention, which also falls within the scope of the invention.

Claims (1)

1. The application of the marker gene miR-429 in downstream gene regulation in sow farrowing potential judgment is characterized in that: the downstream genes are ZEB1 and LH beta; the downstream gene is regulated and controlled by miR-429 to inhibit the expression of a target gene ZEB1, a negative regulation relation is formed between ZEB1 and LH beta gene, and the expression of the LH beta gene is indirectly up-regulated by miR-429 through inhibiting the expression of the target gene ZEB 1; the marker gene miR-429 is positively correlated with the farrowing potential of the sow, the target gene ZEB1 is negatively correlated with the farrowing potential of the sow, and the target gene LH beta is positively correlated with the farrowing potential of the sow;

judging the farrowing potential of the sow by analyzing the expression levels of a marker gene miR-429, a target gene LH beta and a target gene ZEB 1;

the method specifically comprises the following steps: (1) tissue RNA extraction: extracting oviduct and pituitary RNA of different individual sows; (2) designing a primer: respectively designing a q-RT-PCR primer ssc-miR-429 of a candidate gene miR-429, a q-RT-PCR primer ZEB1 of ZEB1 and a q-RT-PCR primer LH beta of LH beta according to a target gene to be analyzed; (3) reverse transcription to synthesize cDNA: synthesizing cDNA (complementary deoxyribonucleic acid) from the total RNA obtained in the reverse transcription step (1) by using a reverse transcription kit; (4) determining the relative expression amounts of the candidate genes miR-429, ZEB1 and LH beta in the oviduct and pituitary of the sow by adopting a q-RT-PCR method; (5) according to the expression level of miR-429 in the oviduct and the pituitary, the expression level of ZEB1 and LH beta in the pituitary; judging the relative high and low farrowing capacity among different sow individuals;

wherein the sequence of the primer ssc-miR-429 is shown as SEQ ID number 1;

the upstream sequence of the primer pair LH beta is shown as SEQ ID number 2, and the downstream sequence is shown as SEQ ID number 3;

the upstream sequence of the primer pair ZEB1 is shown as SEQ ID number 4, and the downstream sequence is shown as SEQ ID number 5;

the step (2) also comprises the design of an internal reference gene primer, wherein the internal reference gene of miR-429 is U6, and the primer sequence is shown as SEQ ID number 6; the internal reference genes of LH beta and ZEB1 are beta-Actin, the sequence of the upstream primer is shown as SEQ ID number 7, and the sequence of the downstream primer is shown as SEQ ID number 8;

performing reverse transcription by using a reverse transcription kit in the step (3), wherein a reverse transcription system is 20 muL, in a miR-429 reverse transcription process system, 1 muL of total RNA, 2 muL of reverse transcriptase mixture, 5X Reaction Mix4 muL of total RNA, and ultrapure water is added to 20 muL; the reaction conditions are as follows: inactivating reverse transcriptase at 37 deg.C for 60min, and maintaining at 95 deg.C for 5 sec;

in a LH beta and ZEB1 reverse transcription process system, 2.0 muL of total RNA, 1 muL of gDNA Eraser, 2.0 muL of 5 XgDNA Eraser Buffer and 20 muL of ultrapure water are added; the reaction conditions are as follows: reacting at 37 ℃ for 15min, and maintaining at 85 ℃ for 5sec to inactivate reverse transcriptase;

the q-RT-PCR method in the step (4) comprises the following reaction system: miR-429 is a 20 muL reaction system, EXPRESS SYBR GreenER ™ qPCRSuperMix Universal green fluorescent dye 10 muL, 2 muL primer ssc-miR-429 and cDNA 2 muL are added with ultrapure water to 20 muL; an LH beta reaction system, 7.5 mu L of iTaq Universal SYBR Green Supermix Green fluorescent dye, 0.5 mu L of each upstream and downstream sequences of a primer pair LH beta, 1.5 mu L of cDNA1.5 mu L and ultrapure water added to 20 uL; the ZEB1 reaction system is the same as LH beta, and the primer pair is ZEB 1;

the reaction process is as follows: miR-429 is performed for 40 cycles of 95 ℃ for 20s, 95 ℃ for 5s and 60 ℃ for 20s, and fluorescence values are collected at 60 ℃ for 20 s; LH beta and ZEB1 are at 95 ℃ for 3min, 95 ℃ for 5s, 60 ℃ for 30s for 40 cycles, and fluorescence values are collected at 60 ℃ for 30 s; wherein the sow breeds are Anhui south black pigs and Yorkshire pigs.

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