CN107058557A - Influence the function candidate gene and its screening technique of pig flesh characters - Google Patents
Influence the function candidate gene and its screening technique of pig flesh characters Download PDFInfo
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Abstract
The function candidate gene and its screening technique of pig flesh characters are influenceed the invention discloses a kind of, wherein function candidate gene is RXRG, GPI, CPT2 and/or GABARAPL1.Screening technique comprises the following steps：(1) longissimus dorsi muscle of two big extreme pig kinds of Meat Quality difference is chosen, DNA is extracted, independently builds storehouse；(2) complete genome DNA is carried out to sample DNA storehouse using MeDIP Seq technologies to methylate sequencing；(3) collection of illustrative plates of the complete genome DNA methylation level of two extreme pig kinds is obtained, and screens differential methylation expressing gene；(4) GO functional annotations and KEGG Pathway are carried out to differential methylation gene to analyze, filters out and the related functional gene and regulatory pathway of Meat Quality such as generate and decompose to aliphatic acid；(5) MeDIP seq and RNA seq result of the test are combined, the Conjoint Analysis of DNA methylation and express spectra is carried out, the function candidate gene of pig flesh characters is obtained.The present invention, which is filtered out, participates in the related function candidate gene of pig flesh characters regulation, is that further investigation from now on lays the foundation.
Description
Technical field
The function candidate gene and its screening technique of pig flesh characters are influenceed the present invention relates to a kind of.
Background technology
Pig flesh characters are the important indicators of the economic protein production of animal.Although having been carried out largely being directed to pork at present
The research of the genetic mechanism of matter character, but full-length genome level explores the research of candidate gene and genetic mechanism or relative has
Limit.The DNA sequencing technology fast development of past 20 years, has become the important means of research genetic mechanism at present.Substantial amounts of research
Show that transcription factor, growth factor receptors and signal path mechanism participate in the regulation process of Meat Quality Traits formation.
DNA methylation is one of gene epigenetic modification for finding earliest, and previous report shows, the CpG methylated
Dinucleotide by change chromatin Structure or influence some transcription factors binding activity suppressor expression.While DNA
Methylate the different physiological processes of regulation, including embryonic development, x x chromosome inactivations, cell differentiation and the table for suppressing imprinted genes
Reach.DNA methylation almost only occurs in the position of 5' carbon cytimidines[8].But proportion is only in genome for these CpG points
For 1%.Far below the frequency of occurrences of other dinucleotides sequences[9].70%~80% CpG dinucleotides are in DNA methylation
State.But there are some regions, the non-CpG methylated is not uniformly distributed, and localized clusters tendency is presented, and is cytimidine
With the enrichment region of guanine, CpG dinucleotide sequences density is very high, using size as 300-3000bp or so and rich in the cores of CpG bis-
The form on the CpG islands of thuja acid is present, and the frequency of occurrences is 5 times of average value, forms CpG islands, CpG islands are typically in the non-shape that methylates
State.Li in 2012 etc. depicts pig genomic methylation profile using MeDIP-Seq methods, explores the different phenotypes of full-length genome
Adipose tissue in relation between fat deposition and DNA methylation.Choi in 2015 etc. using RRBS methods to pig five not
Complete genome DNA is carried out with tissue and cell line to methylate sequencing, is observed and is shown pig full-length genome methylation profiles and people
The mammals such as class have a very high similarity, CpG methylate ratio and gene function organized at five in (remove cell line)
Positive correlation.Test and provide important information for the exploration of pig epigenetics.2006 are waited by gene microarray analysis plan south
Mustard full-length genome methylates, and shows that DNA methylation acts not only on promoter and enhancer and also played the part of in intergenic region and gene regions
Drill key player.Some scholars think that not all gene has identical to methylate and gene expression pattern.Have
Research shows that promoter region CGI missing is the common feature for the miRNA that DNA methylation suppresses.
With being understood in depth to science of heredity and epigenetics, we are directed to screening candidate gene.Choose succulence
Two big extreme pig kinds of shape difference, Wan Nan flower pigs (lard type) and Yorkshire Pigs (lean meat species), be study pig growth performance with
The ideal model of meat differentiation.The purpose of our research is com-parison and analysis Wan Nan flower pigs and Yorkshire Pigs complete genome DNA
Methylation patterns, identification screening may participate in the key gene of fatty acid metabolism and fat deposition process.Utilize MeDIP-seq side
Method explores the contact of DNA methylation and two breeding pig kind meat difference.Obtain hyper-methylation and low first under full-length genome level
Quantity and distribution of the base Peaks and DMRs in gene expression characteristicses region and CGI.In addition, filter out GO functional annotations and
The entry related with fat deposition to fatty acid metabolism and path in KEGG Pathway path analysises, make correlation function and
Path analysis.In combination with transcript profile sequencing, the Conjoint Analysis of DNA methylation and transcript profile express spectra is carried out, is filtered out simultaneously
The gene of differential methylation and differential expression.It is last to select 7 candidate genes progress pyrosequencings, validating DNA methyl at random
Change the accuracy of co-immunoprecipitation.
The content of the invention
The function candidate gene of pig flesh characters is influenceed the technical problem to be solved in the present invention is to provide a kind of.
The invention solves the problems that another technical problem be to provide it is a kind of influence pig flesh characters function candidate gene
Screening technique.
Function candidate gene for influenceing pig flesh characters, the technical solution adopted by the present invention is, function candidate gene
It is RXRG, GPI, CPT2 and/or GABARAPL1.
RXRG, GPI, CPT2 and GABARAPL1 therein nucleotides reference sequences, are surveyed by international pig genome
The pig whole genome sequence that sequence alliance (International Swine Genome Sequencing Consortium) announces
Issue.
For screening technique, the technical solution adopted by the present invention is to comprise the following steps：
(1) longissimus dorsi muscle of two big extreme pig kinds of Meat Quality difference is chosen, DNA is extracted, independently builds storehouse；
(2) co-immunoprecipitation sequencing technologies are methylated to the full base of sample DNA storehouse progress using high-throughout complete genome DNA
Because of a group DNA methylation sequencing；
(3) collection of illustrative plates of the complete genome DNA methylation level of two extreme pig kinds is obtained, and screens differential methylation base
Cause；
(4) GO functional annotations are carried out to differential methylation gene and KEGG Pathway is analyzed, filtered out and given birth to aliphatic acid
Into to the related functional gene and regulatory pathway of Meat Quality such as decompose；
(5) MeDIP-seq and RNA-seq result of the test is combined, the Conjoint Analysis of DNA methylation and express spectra is carried out,
Obtain the function candidate gene of pig flesh characters.
Preferably, in step (1), described two extreme pig kinds are Wan Nan flower pigs and Yorkshire Pigs respectively.
In addition, above-mentioned functions candidate gene is applied in pig flesh characters research.
The beneficial effects of the invention are as follows：
Filter out and participate in the related function candidate gene of pig flesh characters regulation, be that further investigation from now on lays the foundation.
Embodiment
The present embodiment is a kind of screening technique for the function candidate gene for influenceing pig flesh characters, specifically includes following step
Suddenly：
1 materials and methods
1.1 Wan Nan spend the collection of pig and Yorkshire Pigs longissimus dorsi muscle sample
The sample that this experiment is chosen spends pig and Yorkshire Pigs for Wan Nan.Two groups of swinery bodies, every group of 3 individuals and be random
Choose, weight range is between 100-131kg, totally 6 individuals.Take pig body longissimus dorsi muscle meat sample (about 1kg, positioned at last rib
To the 4th rib reciprocal), preserved loaded in cryopreservation tube, being put at once in liquid nitrogen container.Collection is taken out after terminating and is put into -80 DEG C of ice
Case freezes standby.
1.2 longissimus dorsi muscle DNA extraction is built with sequencing library
This experiment uses phenol:Chloroform:Isoamyl alcohol 25:24:1/Agencourt AMPure beads and according to factory
The Standard Operating Procedure that business provides carries out the DNA extractings of sample and purified.DNA is passed throughND-1000 spectrophotometrics
Meter and 0.8% agarose gel electrophoresis quality inspection are qualified rear standby.Sequencing Flow cell used are Illumina SE Flow
Cell v3-HS.Sequencing library, which is built, chooses qualified sample totally 6 groups of making DNA ponds, is respectively designated as WH-1, WH-2, WH-3,
YY-1, YY-2, YY-3, for follow-up MeDIP-seq and checking test pyrosequencing.
1.3 Cluster are generated
It is supporting in the sequenators of Illummina HiSeq 2500 according to corresponding flow shown in cBot User Guide
Cluster generations and first are completed on cBot to sequencing primer to hybridize.
Machine is sequenced on 1.4
Prepare sequencing reagent according to HiSeq 2500User Guide, by machine (instrument on the flowcell for carrying cluster
Model：HiSeq 2500, Illumina).From single-end programs, 1 × 50nt of single end are carried out
Multiplex is sequenced.The data collection software that sequencing procedure is provided by Illumina are controlled, and are carried out
Real-time data analysis.
2 results and analysis
2.1 high flux MeDIP sequencing analysis
Test group WH-1, WH-2 and WH-3 sample generates 33 179 625,37 610 867 and 32 924 118 respectively
Effective reads, test group YY-1, YY-2 and YY-3 sample generates 31634309,31056478 and 27457277 effectively respectively
reads.(table 1) after low-quality initial data is removed, six groups of data generate average 30.8Gb clean reads, effectively
Reads rates are 98.84% to 98.96%.Then all reads are mapped to reference gene group, according to software Sscrofa
10.2 have 77.73% to 85.65% effective percentage.Uniquely mapped reads are that the basis further analyzed is sequenced.
Show that reads is distributed mainly on CDS areas, nearly promoter region transcription initiation site (TSS) upstream 2k regions methylation simultaneously
It is minimum.
The reads quantity of the MeDIP-seq of table 1 sequencing generations
Identifications and certification of 2.2 Peaks in gene region
The peaks quantity of the MeDIP-seq of table 2 sequencing generations
| Sample ID | Peak count | Sample ID | Peak count |
| WH-1 | 120,443 | YY-1 | 194,507 |
| WH-2 | 206,080 | YY-2 | 223,008 |
| WH-3 | 193,398 | YY-3 | 171,769 |
According to the principle of sequencing, MACS (version are used:1.4.2) system, we obtain 1109205 altogether
peaks.The peaks quantity of specific each individual is shown in Table 2.We learn that the peaks numbers scope of 6 samples is 120443 from table
~223008, and in quantity and distribution widely different (table 2).All reference gene group chromosomes are detected, peaks is found
Intergenic region is distributed mainly on, illustrates that intergenic region there may be the potential quality of regulation biological function.Secondly it is introne and CDS areas
Domain, in addition translational termination site downstream 2k, transcription initiation site upstream 2k relatively low (nearly promoter region), 5 '-UTR and 3 '-
UTR is minimum.We have obtained complete genome DNA and methylated Circos displaying figures simultaneously.
Identifications and certification of 2.3 DMRs and DMGs in gene region
Yorkshire Pigs and Wan Nan flower pig complete genome DNA methylation patterns are compared, symbiosis is into 58283 differential methylations
Region, wherein 12086 supermethylations, 46197 hypomethylations (YY VS WH).DMRs is distributed mainly on introne, outer aobvious
Son and CDS regions, translational termination site downstream 2k, transcription initiation site upstream 2k take second place, and 5 '-UTR and 3 '-UTR are minimum.DMRs
Experimental result and report before consistent[23].Previous research defines promoter and gene ontology
Differential methylation gene[24].We know together other 1425 differential methylation genes, wherein 447 up-regulations, 978 downward (YY
VS WH).Wherein 1161 DMGs, which are located at, includes sub-district (387 up-regulations, 779 downwards), and 486 DMGs are located at CDS regions
(47 up-regulations, 439 downwards), 167 DMGs are located at 2kb upstream regions (40 up-regulations, 127 downwards), 229
DMGs is located at 2kb downstream regions (36 up-regulations, 193 downwards), and 116 DMGs are located at 3 ' UTR regions (on 9
Adjust, 107 downwards), 43 DMGs are located at 5 ' UTR regions (7 up-regulations, 36 downwards).It can be seen that hyper-methylation quantity is low
In hypomethylation quantity (YY VS WH).Gene may be simultaneously present in different regions simultaneously.We have obtained entirely simultaneously
Genomic DNA methylation level map, image display DMRs distribution situation.
2.4 clustering thermal maps
In recent years, increasing research confirms that gene expression regulation mechanism is a lot, and DNA methylation is one of them.
We carry out clustering using sequencing data to all samples.First, 6 same kinds of sample flock together, and illustrate same
The methylation of one kind is similar.The methylation of Wan Nan flower pigs is higher than Yorkshire Pigs (table 7A).Show that DNA methylation can
Can influence the latency of Wan Nan flower pigs and Yorkshire Pigs Meat Quality difference.
The promoter region of pig is only about half of to contain CGI.And promoter region CGI DNA methylation is frequent and gene
Transcriptional activity is relevant.In general, methylating for the upper cytimidines of CGI is suppressed by combining transcription factor or changing chromatin Structure
Genetic transcription.Experiment shows that promoter region is consistent with full genome level with the clustering thermal map result in CGI regions.
2.5 DMGs GO functional annotations
Yorkshire Pigs are carried out using the biological Co., Ltd's related software of Shanghai uncle person of outstanding talent and Wan Nan spends the differential methylation base of pig
Because of biological analysis.GO functional annotations are related to 346 functional category (P altogether<0.05), comprising three aspects.Wherein cellular component
40 entries, the 11.56% of accounting example, 54 entries of molecular function, the 15.61% of accounting example, 252 bars of biological function
Mesh, the 72.83% of accounting example.There are some to be synthesized on fatty acid metabolism process, fatty acid biological in biological function entry
Journey, Regulating Lipid Metabolism, cytolipin metabolic process and metabolism of organic acids process (table 3).In addition, we are for methylating
The upper situation for reconciling downward makes before ranking 30 biological function displaying figure (P respectively<0.05).
The Yorkshire Pigs of table 3 and Wan Nan flower pig longissimus dorsi muscle differential methylation gene GO functional annotations
2.6 DMGs KEGG Pathway path analysises
Yorkshire Pigs are carried out using the biological Co., Ltd's related software of Shanghai uncle person of outstanding talent and Wan Nan spends the differential methylation base of pig
Because of the prediction of KEGG Pathway path analysises.Experimental result shows differential methylation gene significant enrichment in 277 pathways
In network path, wherein 24 may be related to meat.Including PPAR signal paths, Adipocyte Factor signal path, AMPK
Signal path and the lipolysis of regulation and control fat cell etc..It is polygenes, a Multi net voting to further disclose pig flesh characters
The character (table 4) of regulation and control.In addition, we are directed to the upper situation about lowering that reconciles that methylates and made respectively 30 before ranking
KEGGPathway path analysises displaying figure (P<0.05).
The Yorkshire Pigs of table 4 and Wan Nan flower pig longissimus dorsi muscle differential methylation gene KEGG Pathway path analysises
2.7 MeDIP-Seq and RNA-Seq Conjoint Analysis
DNA methylation is occurred mainly near the CGI of gene promoter area, and methylating for CGI will be significantly inhibited or complete
The transcription of full cryptiogene, and then influence the generation of protein.RNA-seq can accurately measure differential expression by setup parameter
Gene, and the relation negatively correlated with transcript profile that methylate.Therefore, we combine DNA methylation and transcript profile data are entered
Row association analysis, explores the relation that Yorkshire Pigs and Wan Nan flower pig DNAs methylate between transcript profile.
The Conjoint Analysis of Yorkshire Pigs and Wan Nan flower pig longissimus dorsi muscle DNA methylations and transcript profile, we recognize high methyl
Change the gene of correspondence low expression high expression corresponding with hypomethylation, its methylation is opposite with the situation of transcription.It is existing to grind
Study carefully and show that these genes participate in the genetic mechanism related to meat, it is the property that polygenes regulates and controls to further disclose Meat Quality
Shape.
The other differential methylation gene 1425 of experiment common recognition, difference expression gene 347.Conjoint Analysis shows there are 80 bases
Because there is negative correlativing relation, wherein RXRG, GPI, CPT2 and GABARAPL1 gene is significantly negatively correlated.
Associating summary table is：correlation_all.txt.Directly draw and obtain correlation_all.pdf, but this
Figure can not intuitively embody their direct correlations very much, so being adjusted according to the result of figure.According to figure, (reference axis is -3
Gene is more in the range of to 3), screen x, y-axis.Further screen y-axis：log(FoldChange_YY.WH)>Or log 0.5)
(FoldChange_YY.WH)<-0.5.The contingency table of screening is：correlation_filter05.txt.Gene after screening
Color-coded again and (calculating gene number) just obtains plus mark：Correlation_filter05.pdf notes：The base repeated
Because being screened according to FoldChange_YY.WH scores.The form that screening significant difference is obtained：second_
Correlation.txt, the log (FoldChange_YY.WH) of the quadrant of figure second>1 and edgeR.logFC<-1；fourth_
Correlation.txt, the log (FoldChange_YY.WH) of figure fourth quadrant<- 1 and edgeR.logFC>1.
2.8 pyrosequencing
Randomly select 7 differential methylation genes and carry out pyrosequencing certification, it was demonstrated that DNA methylation co-immunoprecipitation
Accuracy.7 genes are respectively ACOX1, GABARAPL1, LEP, PPARD, GNB3, APOC3, NR6A1.pyrosequencing
As a result consistent with MeDIP-seq results and significant difference (P<0.05) it is accurately may be used, to show that MeDIP-Seq sequencings obtain result
Lean on.
3 discuss
This experiment spends pig and Yorkshire Pigs longissimus dorsi muscle as sample using Wan Nan, draws out a comprehensive complete genome DNA
Methylation patterns figure.Previous research has confirmed that methylating for promoter region is an important epigenetic regulation
The expression of mechanism and cryptiogene.Our experimental verification this point, and confirm the methylation level phase of promoter region
Other regions are kept with relatively low state, and (define gene ontology is gene ontology/higher state of protein coding region holding
Gene transcription start site is to the part of translational termination site).Methylating for gene ontology is believed while being recognized as a kind of suppression
The extension of transcription number is hindered, and is verified in arabidopsis and mammalian cell.Meanwhile, have article report Hongyuan chicken and
AA broiler chicken liver and musculature complete genome DNA methylate research, in nearly promoter region, the expression of gene and
DNA methylation level is negatively correlated.
Wan Nan spends the comparison of pig and Yorkshire Pigs full-length genome methylation profiles to disclose the process of muscle differentiation, illustrates
Different DNA methylation ideographs.The article reported before is consistent, includes some significant biological pathways.Including PPAR
Signal path, MAPK signal paths, Adipocyte Factor signal path, Wnt signal paths, insulin signaling pathway and regulation and control fat
The lipolysis of fat cell.Wherein, PPAR signal paths are logical on muscle differentiation and the most important signal of meat forming process
One of road.PPAR signal paths as fatty sensor adjustment fatty acid metabolism enzyme transcription.There are PPAR α, PPAR β and PPAR γ
Three kinds of hypotypes.PPAR α are concentrated mainly in the higher tissue of fatty acid metabolism, such as liver, brown fat, heart, kidney and bone
The expression of bone flesh.PPAR α include energetic supersession by many physiological functions of the Expression modulation for adjusting target gene, grown simultaneously
Deng.In addition, it also adjusts cell growth, differentiation and apoptosis by the biological acceptor of regulating lipid metabolism.PPAR β wide expressions in
Various Tissues, PPAR γ adjust the differentiation of fat cell, and important function has been played in glycometabolism.PPARs combination RXRs acceptors
Transcription activity could be completed.By Wan Nan is spent pig and Yorkshire Pigs Meat Quality Traits DNA methylation Pathway conspicuousnesses
Enrichment analysis, it was demonstrated that DNA methylation is probably to cause the major reason of muscle differentiation.
Analyzed with reference to GO functional analyses and Pathway paths significant enrichment, it is related that we have screened several Meat Quality Traits
Gene, including ACOX1, APOC3, LEP, PPARD, ADIPOQ, RXRG, GPI, CPT2, CROT, AKT2 etc..
Acyl coenzyme A oxidizing ferment 1 (ACOX1) is a member of ACOX gene families, is primarily involved in fat in peroxisome
The oxidation decomposition course of fat acid.ACOX1 is the rate-limiting enzyme of dehydrogenation reaction, take part in the sour beta-oxidation first of peroxisomal fatty
Step.ACOX1 enzymes are extremely conservative simultaneously, possess relatively unique expression way, transcription generation mRNA and its protein expression master
Liver, kidney are distributed in, next to that brain and adipose tissue.Fully prove ACOX1 genes in fat deposition and fat metabolism
Aspect plays potential effect.Meanwhile, the report before many also demonstrates that ACOX1 genes in daily gain, birth weight, the thickness of backfat
With aliphatic acid composition etc. these important quantitative inheritance characters play a significant role.Our experiences show that ACOX1 genes are being opened
Sub-area, Yorkshire Pigs methylation level spends the Wan Nan flower pigs of pig, i.e. hypomethylation level to promote ACOX1 table higher than Wan Nan
Reach.
ApoC 3 (APOC3) gene is located at No. 9 chromosomes.It is considered as the horizontal shadow by adjusting triglycerides (TG)
Ring fat metabolism and deposition.Favour et al. have studied the expression of APOC3 in seven tissues of laughable pig and Jiangkou radish pig, wherein
Expression quantity highest is in liver, next to that hypodermis and longissimus dorsi muscle.APOC3 is to constitute HDL-C
(HDL) and the rich ester lipoprotein (TRL) of glycerine 3 important gene, in liver and small intestine synthesis APOC3, and then it is related to suppress lipid
The activity of metabolic enzyme, blocks corresponding acceptor in liver to be combined with enzyme, so as to influence the metabolism of lipid.
LPIN1 genes influence the synthesis of fat in terms of two.On the one hand, LPIN1 gene codes phosphatidic acid phosphatidase, ginseng
With the synthesis of phosphatide and triglyceride.On the other hand, adjust Adipocyte Differentiation, formed as activity factor induced lipolysis.
Leptin genes are a kind of proteohormones encoded by leptin gene, and it secretes output by fat cell, form a kind of molecule
Measure the protein hormone for 16ku.Have in numerous physiological activities such as fat deposition, energetic supersession, body weight growth important
Adjustment effect.Et al. DNA methylation transcription analysis, inspection are carried out to the CpG sites of five particular organization's promoter regions of fat
The expression in different tissues mRNA has been surveyed, has as a result shown the expression degree highest of leptin.Acetyl coenzyme A (ACACB,
Acetyl-Co A carboxylase beta) it is the differential methylation expressing gene that testing sieve is selected, synthesize in the cell, together
When be also fatty acid synthesis pathway rate-limiting enzyme, be fat cell obtain aliphatic acid another important channel.Carnitine O octyl groups
Transferase (CROT) is a kind of peroxidase, by coupling short chain fatty acids formation carnitine into mitochondrial matrix and combining
Carnitine palmitoyltransferase Ⅰ (CPT1A) carries out the oxidation of aliphatic acid and adjusts the metabolism of aliphatic acid.Stearoyl-Co A dehydrogenations
Enzyme (Stearoyl-CoA desaturase, SCD) is a kind of can to catalyze and synthesize the iron enzyme of monounsaturated fatty acids.Mainly
It is present in mammalian fat tissue and mammary gland.Research shows that SCD can carry out metabolic regulation to skeletal muscle, simultaneously
New life of ceramide etc. in insulin sensitivity and mitochondrial fatty acid oxidase, oxidation muscle fibre can be influenceed.In addition,
SCD is also the important modifying ingredients of leptin signaling pathway, is one of most important leptin regulatory gene, may have one to leptin
Fixed downward effect.GPI genes play a significant role in cell glycolysis and gluconeogenesis.How people's research confirms GPI not only
Signal transduction can be participated in, and participates in some receptors signal transductions of " tradition ".So it be able to may be led to by coherent signal
Road adjusts glycolysis and gluconeogenesis.AKT2 genes, which can be mediated, adjusts different growth factor and hormone, participate in boar into
The regulation process such as long, development, metabolism.
This seminar RNA-seq result of the test, carries out DNA methylation and expression with reference to this MeDIP-seq and before
The Conjoint Analysis of spectrum, screen existing methylation differential has the gene of differential expression again.As a result show, Yorkshire Pigs and Wan Nan flower pigs
Compared to having 4 differential methylations and expressing gene (P<0.05).
In four genes, RXRG genes are a genes related to porcine intramuscular fat deposition, positioned at No. 4 chromosomes
On.RXRG targets gene, and then regulating cell differentiation and histomorphological changes by combining particular responses region.RXRG is participated in
PPAR signal paths, are a kind of nuclear receptor proteins, in cell differentiation, development and metabolism (carbohydrate, lipid, protein) hair
Wave important function.In our experiment, Wan Nan flower pig RXRG genes show as hypomethylation, but RXRG genes are shown as
The overexpression of gene.Methylating for RXRG genes, to a certain extent, may participate in Meat Quality regulation, such as fat deposition, fat
Fat acid metabolic and biosynthesis, the regulation and control such as Adipocyte Differentiation.Hale in 2006 analyzes zebra fish RARG gene pairs embryo hair
The influence educated.
GPI genes be before the difference expression gene related to Meat Quality reported.In cell glycolysis and sugar
Played an important role in heteroplasia.He in 2012 research shows that GPI can not only participate in signal transduction, while also participating in some " pass
The signal transduction of the acceptor of system ".Therefore it may adjust glycolysis and gluconeogenesis by associated signal paths.Our experiment knot
Fruit shows that the possibility of Wan Nan flower pig GPI gene hypomethylation levels influences Meat Quality.
CPT2 is the key gene in fatty acid metabolism approach, relies on the fatty acid oxidation that its crucial rate-limiting enzyme is participated in.
CPT2 can also adjust the decomposition and energy supply of aliphatic acid.Meanwhile, CPT2 is also by various cell factors, such as malonyl-CoA
Regulation.Either before transcription, translation or post-transcriptional level, all showing internal fat accumulation is strictly regulated and controled by CPT2.
It was reported that, in pork, chicken, addition l-cn can help improve meat in the feed such as fish, improve local flavor.CPT2 is in pig
The gene that body is significantly expressed.It is distributed mainly on heart, liver, spleen, skeletal muscle etc..We ground at experimental result with former
Study carefully consistent.CPT genes play a significant role during label of pig fat deposition description, and have not in musculature and adipose tissue
Same expression pattern.This is consistent with our result.
GABARAPL1 is a member of GABARAP families.There are some domains by this in Liu Faxian GABARAPL1 in 2014
The upstream insulin signaling regulation of gene promoter area, referred to as insulin replies element (IRE).And confirm that insulin is
The most important regulatory factor of GABARAPL1 transcriptional level.Research shows that the potentiality (GP) of GABARAPL1 and glycolysis have
Close, and adjusted by hormonal readiness, such as insulin, hyperglycemic factor and adrenaline.Simultaneously with various Meat Qualities
Also it is closely related.Physiological and biochemical index and muscle Meat Quality correlation analysis show that the influence that GP is formed to final meat is limited.
The report insulin such as Liu can suppress mouse primary hepatocytes GABARAPL1 expression, and the increase of this expression can be with
Suppressed by insulin inhibitor LY294002.In our experiment, GABARAPL1 is supermethylation and low in Wan Nan flower pigs
The gene of expression, GABARAPL1 genes may influence Meat Quality indirectly by the regulation and control of insulin signaling.
4 conclusions
This experiment depicts complete genome DNA methylation profiles, compares Yorkshire Pigs and Wan Nan flower pig longissimus dorsi muscles DNA
The influence methylated to meat quality.Know together other 1425 differential methylation genes.DNA methylation and the joint of transcript profile sequencing
Analysis confirms the expression for the influence gene that methylates, and obtains function candidate gene RXRG, GPI, CPT2 of 4 pig flesh characters
And/or GABARAPL1.Candidate gene and GO functional annotations, the table that KEGG Pathway path analysises are pig from now on filtered out
See science of heredity and one good reference role of offer is provided.
The embodiments of the present invention described above are not intended to limit the scope of the present invention.It is any in the present invention
Spirit and principle within the modifications, equivalent substitutions and improvements made etc., should be included in the claim protection model of the present invention
Within enclosing.
Claims (4)
1. influence the function candidate gene of pig flesh characters, it is characterised in that function candidate's base of the influence pig flesh characters
Because being RXRG, GPI, CPT2 and/or GABARAPL1.
2. influenceing the screening technique of the function candidate gene of pig flesh characters, comprise the following steps：
(1) longissimus dorsi muscle of two big extreme pig kinds of Meat Quality difference is chosen, DNA is extracted, independently builds storehouse；
(2) co-immunoprecipitation sequencing technologies are methylated to sample DNA storehouse progress full-length genome using high-throughout complete genome DNA
DNA methylation is sequenced；
(3) collection of illustrative plates of the complete genome DNA methylation level of two extreme pig kinds is obtained, and screens differential methylation gene；
(4) GO functional annotations and KEGG Pathway is carried out to differential methylation gene to analyze, filter out with aliphatic acid generation and
The Meat Qualities such as decomposition related functional gene and regulatory pathway；
(5) MeDIP-seq and RNA-seq result of the test is combined, the Conjoint Analysis of DNA methylation and express spectra is carried out, obtained
The function candidate gene of pig flesh characters.
3. screening technique according to claim 2, it is characterised in that in step (1), described two extreme pig kinds are respectively
Wan Nan spends pig and Yorkshire Pigs.
4. application of the function candidate gene of influence pig flesh characters in pig flesh characters research as claimed in claim 1.
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