CN107904242B

CN107904242B - The new lncRNA of one boar intramuscular fat tissue extraction and its application

Info

Publication number: CN107904242B
Application number: CN201711461874.7A
Authority: CN
Inventors: 苗向阳; 黄万龙; 张秀秀; 李嫒; 解领丽
Original assignee: Institute of Animal Science of CAAS
Current assignee: Institute of Animal Science of CAAS
Priority date: 2017-12-28
Filing date: 2017-12-28
Publication date: 2019-11-05
Anticipated expiration: 2037-12-28
Also published as: CN107904242A

Abstract

The invention discloses the new lncRNA of a boar intramuscular fat tissue extraction and its applications.Invention RNA-seq technology and bioinformatics method, comparative analysis Large White and Laiwu Pigs intramuscular fat Gene Expression Profiles, evaluation and screening at rouge differentiation and the relevant key difference expression lncRNA of lipid metaboli.Molecular experiment display lncRNA and its target gene PLEKHG3, SERPINB7, SPTLC3, TMOD2 are closely related with pig intramuscular fat.The present invention is that the lipidosis mechanism study of pig and domestic animal adipose tissue lncRNA study based theoretical, has important application prospect in domestic animal molecular breeding.

Description

The new lncRNA of one boar intramuscular fat tissue extraction and its application

Technical field

The invention belongs to technical field of molecular biology, and in particular to the new lncRNA of a boar intramuscular fat tissue extraction And its application, it more particularly relates to XLOC_007365 and is applied in breeding high-quality domestic animal kind.

Background technique

Adipose tissue has great influence, especially intramuscular fat to meat, including the appearance of meat, flavor, holds Aqueous, tenderness etc., however, during pursuing the long-term breeding of high lean meat percentage, so that intramuscular fat content reduces in pig carcass, Meat quality decline.Adipose tissue is the tissue of a storage energy simultaneously, human fatty tissue over-deposit, cause it is fat and Energy metabolism balance imbalance, obesity-related disorders can be caused, as type II diabetes, insulin resistance, cardiovascular disease and certain A little cancers, seriously threaten human health.Therefore more and more attention has been paid to grind human health problems caused by meat and obesity It is diseases related to fat is treated and prevented to study carefully fat deposition molecular mechanism, and improves meat and is of great significance.

Laiwu Pigs have the valuable germplasm such as reproductive capacity is high, meat is excellent special as the excellent local pig breed resource in one, China Property, it is the Typical Representative of the black pig in Shandong Province place, content of fat in body is relatively high, and muscle has scarlet yellowish pink, good Water retention property, more importantly containing more rich intramuscular fat (10.32%).However, Large White comes as China's pork One principal item in source has high feed conversion rate and dressing percentage and stronger adaptability, is typical bacon hogs kind, The subcutaneous and intramuscular fat content that it is deposited is less.The especially deposition of intramuscular fat, compared to China's local varieties, such as two , there is significant difference in paint face, Laiwu Pigs and Lu Laihei pig, Laiwu Pigs and Large White are lipidosis and fat cell into rouge point Change provides good experimental material.

LncRNA is that a kind of length is greater than 200 nucleotide, the RNA without encoding histone ability.As a kind of regulation Property non-coding RNA, there is important regulating and controlling effect to lard fat metabolism and at rouge differentiation, while regulating and controlling fatty generation about lncRNA The research for thanking to related disease is also more and more, however there are the different cultivars pig intramuscular fat groups of significant difference for fat deposition The express spectra and function for knitting middle lncRNA rarely have research.

Therefore, it is experimental material that Laiwu Pigs and Large White are chosen in this research, utilizes RNA-seq technology and bioinformatics side Method, comparative analysis Large White and Laiwu Pigs intramuscular fat Gene Expression Profiles, evaluation and screening at rouge differentiation and lipid metaboli phase The key difference of pass expresses lncRNA (XLOC_007365), probes into its molecular mechanism for regulating and controlling porcine intramuscular fat deposition, is pig Lipidosis mechanism study and domestic animal adipose tissue lncRNA study based theoretical, it is intended to be regulated and controled with fat metabolism, for training It educates high meat domestic animal kind and treats and prevents the research of lipid metaboli related disease and provide a certain basis, in domestic animal molecular breeding With important application prospect.

Summary of the invention

The purpose of the present invention is to provide a kind of lncRNA relevant to pig intramuscular fat, entitled XLOC_007365, sequences Column have 90% or more sequence homology with SEQ ID NO.1.

Preferably, XLOC_007365 sequence and SEQ ID NO.1 have 95% or more sequence homology；It is furthermore preferred that long Chain non-coding RNA sequence is SEQ ID NO.1.

The purpose of the present invention is to provide a kind of reagent for detecting pig intramuscular fat, reagent is miscellaneous by sequencing technologies, nucleic acid The expression of friendship technology or nucleic acid amplification technologies detection XLOC_007365.

Preferably, pass through high throughput sequencing technologies, probe hybridization technique, biochip technology or fluorescent quantitative PCR technique Detect the expression of XLOC_007365.

Further, the nucleic acid amplification technologies are selected from polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and be based on nucleic acid sequence The amplification (NASBA) of column.Wherein, PCR needs directly expand RNA reverse transcription at DNA (RT-PCR), TMA and NASBA before amplification Increase RNA.

The purpose of the present invention is to provide a kind of reagent for detecting pig intramuscular fat, reagent includes a pair of for nucleic acid amplification Primer, sequence be SEQ ID NO.2 and SEQ ID NO.3.

Further, the sample of the reagent detection of above-mentioned detection pig intramuscular fat is tissue, preferably intramuscular fat tissue.

The purpose of the present invention is to provide following any one applications:

Above-mentioned lncRNA is predicting or is assisting the application in prediction meat quality；

Application of the above-mentioned lncRNA in preparation prediction or auxiliary prediction meat quality reagent；

Above-mentioned lncRNA has the application in different meat quality pigs in breeding.

The application in prediction meat quality is being predicted or assisted to mentioned reagent；

Application of the mentioned reagent in preparation prediction or auxiliary prediction meat quality reagent；

Mentioned reagent has the application in different meat quality pigs in breeding；

The purpose of the present invention is to provide a kind of reagent for detecting pig intramuscular fat, reagent is miscellaneous by sequencing technologies, nucleic acid The expression of the target gene of friendship technology, nucleic acid amplification technologies or the method for immunoassays detection XLOC_007365, target gene are PLEKHG3 and/or SERPINB7 and/or SPTLC3 and/or TMOD2.

Further, the reagent includes a pair of for expanding the primer of the target gene of XLOC_007365, primer sequence choosing From: the primer pair being made of SEQ ID NO.4 and SEQ ID NO.5；Drawn by what SEQ ID NO.6 and SEQ ID NO.7 was formed Object pair；The primer pair being made of SEQ ID NO.8 and SEQ ID NO.9；It is made of SEQ ID NO.10 and SEQ ID NO.11 Primer pair.

Mentioned reagent has the application in different meat quality boars in breeding.

It would be recognized by those skilled in the art that practicability of the invention is not limited to any spy to XLOC_007365 The gene expression for determining variant is quantified.In some embodiments, have identical as XLOC_007365 sequence at least 85% Or similar cDNA sequence, such as above-mentioned listed sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% the same or similar cDNA sequence.

Term " homologous " be primarily referred to as it is homologous in sequence, that is, be used to illustrate two or more protein or DNA Sequence ancestors having the same.Homologous sequence generally has similar function.The homology of protein and DNA are often through them The similitude of sequence determines, similitude refer to be used to describe during sequence alignment it is between detection sequence and target sequence identical The height of DNA base or amino acid residue sequence proportion.In general, when similarity degree is higher than 50%, often speculate inspection Sequencing column and target sequence may be homologous sequence；When degree of similarity is lower than 20%, just it is difficult to determine if to have same Source property.

In general, PCR uses the annealing of denaturation, primer pair and opposite strand and multiple circulations of primer extend, with index side The copy number of formula increase target nucleic acid sequence；Reverse transcriptase (RT) is then used for the DNA (cDNA) complementary from mRNA preparation by RT-PCR, Then cDNA is generated to multiple copies of DNA by PCR amplification；TMA is in substantially constant temperature, ionic strength and pH Under the conditions of autocatalytically synthesize multiple copies of target nucleic acid sequence, wherein multiple RNA copy of target sequence is autocatalytically given birth to At other copy, TMA is optionally included using blocking, part, terminate part and other modified parts, to improve TMA process Sensitivity and accuracy；LCR uses the two groups of complementary DNA oligonucleotides hybridized with the adjacent area of target nucleic acid.DNA few nucleosides Acid is covalently attached in thermal denaturation, hybridization and the multiple circulations of the repetition of connection by DNA ligase, to generate detectable double-strand Connect oligonucleotide product；SDA uses multiple circulations of following steps: primer sequence pair and the opposite strand of target sequence move back Fire carries out primer extend under there are dNTP α S to generate (hemiphosphorothioated) of half thiophosphorylation of double-strand Primer extension product, the nicking that the endonuclease that semi-modified restriction enzyme enzyme recognition site carries out mediates, and from cutting The polymerase-mediated primer extend that the mouth end 3' carries out is to replace existing chain and generate for next round primer annealing, nicking and displacement Chain, so as to cause product geometry expand.

In the present invention " probe " refer to can be with the molecule in conjunction with the particular sequence of another molecule or subsequence or other parts.It removes Non- to indicate otherwise, term " probe " is often referred to match and another polynucleotides (often referred to as " target multicore glycosides by complementary base Acid ") combine polynucleotide probes.According to the preciseness of hybridization conditions, probe energy and with the probe lack sufficient sequence it is complementary Property target polynucleotide combine.Probe can make direct or indirect label, and range includes primer.Crossing system, including, but not It is limited to: solution phase, solid phase, mixed phase or in situ hybridization measuring method.

The probe has the base sequence with the specific base sequence complementary of target gene.Here, so-called " complementation ", As long as hybridization, can not be complete complementary.These polynucleotides have usually relative to the specific base sequence 80% or more, preferably 90% or more, more preferable 95% or more, particularly preferred 100% homology.These probes can be DNA, It is also possible to RNA, furthermore it is possible to pass through PNA (Polyamide nucleic in part of it or complete nucleotide Acid, peptide nucleic acid), LNA (registered trademark, locked nucleic acid, Bridged Nucleic Acid, Cross-linked core Acid), ENA (registered trademark, 2 '-O, 4 '-C-Ethylene-bridged nucleic acids), GNA (Glycerol Nucleic acid, glycerol nucleic acid), the artificial replacement nucleic acid such as TNA (Threose nucleic acid, threose nucleic acid) obtains Polynucleotides.

Term " hybridization " in the present invention is used to refer to the pairing of complementary nucleic acid.Hybridization and intensity for hybridization are (that is, between nucleic acid Association intensity) influenced by factor such as below: the stringency of complementarity, related condition between nucleic acid is formed Hybrid Tm and nucleic acid in G:C ratio.The individual molecule of pairing in its structure containing complementary nucleic acid is known as " self Hybridization ".

Nucleic acid hybridization technique in the present invention include but is not limited in situ hybridization (ISH), microarray and Southern or Northern trace.In situ hybridization (ISH) be it is a kind of use label complementary DNA or RNA chain as probe with position tissue one Part or slice (original position) or if organize it is sufficiently small if for entirely organize (full organization embedding ISH) in specific DNA or The hybridization of RNA sequence.DNA ISH can be used for determining the structure of chromosome.RNA ISH is for measuring with position tissue slice or entirely MRNA and other transcripts (for example, ncRNA) in organization embedding.Usually sample cell and tissue are handled in situ solid Targeting transcript, and increase the entrance of probe.Probe hybridizes with target sequence at high temperature, then washes off extra probe.Point Not Shi Yong autoradiograph, fluorescence microscopy or immunohistochemistry, in tissue with radiation, fluorescence or antigenic mark base The probe of label is positioned and is quantified.ISH can also be used two or more to pass through radioactivity or other nonradioactive labelings The probe of substance markers, to detect two or more transcripts simultaneously.

Detailed description of the invention

Fig. 1 is intramuscular fat difference expression gene distribution map；

Fig. 2 is difference expression gene qRT-PCR verification result figure；

Fig. 3 is the qRT-PCR verification result figure of difference expression gene XLOC_007365；

Fig. 4 is the target gene qRT-PCR verification result figure of XLOC_007365.

Specific embodiment

The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip Part, or according to the normal condition proposed by manufacturer.

1 sample of embodiment acquisition preparation and experimental design

After experiment pig is butchered, its longissimus dorsi muscle intramuscular fat tissue is acquired rapidly, is cut into small pieces, is packed into 5mL and freezes Guan Zhong puts into liquid nitrogen frozen, is transferred to -80 DEG C of refrigerator long-term preservations later, for the extraction of total serum IgE, 3 groups of experimental setup, divides It is other that Large White intramuscular fat tissue (D_JN) and the lncRNA in Laiwu Pigs intramuscular fat tissue (L_JN) are identified, and point Their intramuscular fat Gene Expression Profiles are analysed, 3 repetitions are arranged in each sample.

The extraction and Quality Control of 2 sample total serum IgE of embodiment

The adipose tissue sample for taking out equivalent cryo-conservation respectively uses mirVana according to operation instructions^TMRNA takes out Extraction reagent kit extracts the total serum IgE of each adipose tissue sample, and isolated total serum IgE sample is stored in -80 DEG C of refrigerators.Using The concentration and OD260nm/OD280nm value of 2000 spectrophotometric determination RNA sample of NanoDrop, and control 1.9~ Between 2.1, using Bioanalyzer 2100 assess total serum IgE quality, and control RIN >=7 and 28S/18S >=0.7, utilize RNase-free DNase I eliminates potential contaminating genomic DNA.

Embodiment 3cDNA library construction and RNA sequencing

Chain specific cDNA libraries

(1) Ribo-zero kit removes rRNA

(2) RNA fragmentation

(3) double-strand cDNA synthesis and purifying

(4) end is repaired, and A base is added

(5) sequence measuring joints connect

(6) DNA fragmentation enriching and purifying

(7) library quality inspection

(8) this research is built together vertical 6 cDNA libraries, respectively D_JN_1, D_JN_2, D_JN_3 (Large White intramuscular fat Tissue cDNA library) and L_JN_1, L_JN_2, L_JN_3 (Laiwu Pigs intramuscular fat tissue cDNA library).

RNA-Seq(Illumina Sequence)

After library quality inspection is qualified, using 2500 microarray dataset of Illumina HiSeqTM, it is sequenced using both-end (Paired-end Sequence) carries out sequencing analysis to cDNA library, and lower machine data are raw sequencing data raw reads.

4 initial data Quality Control of embodiment and filtering

Raw sequencing data (raw reads), there are low quality and contaminated sequence, it is necessary to by quality control and mistake Filter, just can be carried out subsequent bioinformatic analysis process, guarantees the accuracy and reliability of result.Mainly apply cutadapt (v1.12) and FASTX_toolkit (v0.0.14) software carries out quality control to raw reads, and subsequent analysis is based on To clean reads.Concrete operations are as follows:

(1) reads of the removal with the pollution of connector (adapter) sequence；

(2) reads that can not determine that base (N) ratio is greater than 10% in sequence is filtered out；

(3) base for removing mass value Q < 20 accounts for the low quality reads that sequence total bases are greater than 15%；

It the results are shown in Table lattice 1, about 90,000,000 clean reads, and reads obtained by Quality Control, in each sample The base ratio of middle Q-score >=30 is about 95%, while GC base contents account for about 50%, show that sequencing data result can It leans on, can be used for further analyzing after Quality Control.

1 Raw data quality control result of table

Embodiment 5 is spliced with reference to genome alignment and transcript

Clean reads is compared to reference genome, reads is positioned.It is downloaded first from Ensembl database The reference genome Sscrofa10.2 (ftp: //ftp.ensembl.org/pub/release-87/fasta/sus_ of pig ) and comment file Sscrofa10.2.87.chr.gtf (ftp: //ftp.ensembl.org/pub/ scrofa/dna/ release-87/gtf/sus_scrofa).Then bowtie software (v2.2.5) (Langmead&Salzberg, 2012) is used Bowtie-build, which is established, refers to gene group index, with TopHat (v2.0.12) (Trapnell et al., 2009；Kim Et al., 2013) the clean reads that software obtains each sample is compared onto reference genome, and mismatch is limited to 2, other selection default parameters.

In order to predict new transcript, need to rebuild transcript and assembled.With TopHat2 software by clean Sequence alignment file accepted_hit.bam (the resulting that reads is obtained after comparing to genome Alignment files) be input, using Cufflinks (v2.1.1) (Trapnell et al., 2012；Trapnell et Al., 2010) software carries out transcript assembling to each sample, obtains transcript.gtf comment file.It utilizes Cuffmerge assembles the gtf file of 12 samples, merges and generates merged_transcript.gtf comment file.It utilizes Cuffcompare by merged_transcript.gtf and with reference to comment file Sscrofa10.2.87.chr.gtf carry out by One compares, the exact matching such as screening and other known ncRNA, mRNA or similar transcript, while expliciting the position transcript Location information, the potential new mRNA and lncRNA of identification prediction.

As a result: Clean reads being compared into the reference genome to pig using bioinformatics software, as a result such as 2 institute of table Show.

2 Clean reads of table, which is compared, refers to genome result

The analysis of 6 alternative splicing events of embodiment

The assembling file of each sample is analyzed using ASprofile (v1.0) (Florea et al., 2013) software, it is right Variable sheer event carries out statistic of classification.It is detained situation according to the structure of exon and introne, by variable sheer event (alternative splicing, AS) is defined as 12 different classifications, including TSS, TTS, SKIP, XSKIP, MSKIP, XMSKIP、IR、XIR、MIR、XMIR、AE、XAE。

The potential lncRNA of embodiment 7 excavates identification

LncRNA is that a kind of length is greater than 200bp, and the RNA of coding protein, is not based on the two main features, to latent LncRNA identified, between main screening-gene between lncRNA (intergenic lncRNA, lincRNA), introne LncRNA (intronic lncRNA), justice lncRNA (sense lncRNA) and antisense lncRNA (antisense lncRNA).Concrete operations are as follows:

(1) exon number and the screening of transcript length: threshold value is exon several >=2, length > 200bp, is filtered out low Single exon transcript of confidence level.

(2) for the transcript screened above, PLEK (Li et al., 2014), CNCI coding potential screening: are utilized (Sun et al., 2013b), CPC (Kong et al., 2007), Pfam (Finn et al., 2014) these four software predictions Its encoding histone potential, takes intersection to obtain the final result of lncRNA.PLEK is to be based on optimization k-mer strategy, threshold value score < 0, CNCI is based on sequence adjacent nucleotide triplet frequency spectrum, and threshold value score < 0, it is special that CPC is based on transcript open reading frame sequence Sign, and compared with UniProt reference database BLASTX, threshold value score < 0, Pfam are a protein family databases, will be turned Record this encoder block it is homologous compare compare to database less than transcript be lncRNA.

(3) identification of lncRNA known to, ALDB (ADomestic-Animal Long Noncoding RNA Database) (Li et al., 2015a) is a livestock animals lncRNA database, will be candidate by BLASTN tool LncRNA is compared with the lncRNA in database, with Identity=100%, mismatch=0, E-value < 1e-10, gap_ Opening=0 is that condition strictly identifies known lncRNA.

Mainly the classification of lncRNA, distribution of lengths and exon number are analyzed, at the same with identification obtain known to MRNA is compared analysis.Than more consistent, shorter mRNA turns the length of lncRNA and encoding egg white gene generally distribution trend It records this density and is relatively higher than lncRNA, the lncRNA average length identified in this research is 2263nt, and mRNA average length is 2028nt。

The different sample room analysis of gene differential expression of embodiment 8

Known mRNA, the new transcript of prediction and lncRNA data set are constructed, is compared and is united using bowtie and eXpress software Meter analyzes gene expression abundance (read count) of each transcript in each sample.Using in every million segment come from a certain gene Table of segment number (fragments Per kb per Million reads, the FPKM) algorithm of every kilobase length to gene It is corrected, is eliminated because of sequencing depth, the influence that mrna length is different and differences between samples are to gene expression amount up to level.Experiment tool There is biology repetition, using R language pack DESeq2 (Anders&Huber, 2010), negative binomial distribution is based on, to different sample rooms Gene (including lncRNA, mRNA) carries out Differential expression analysis, and Benjamini-Hochberg algorithm is used to carry out P value multiple Hypothesis testing correction, obtains correction P value (padj), with | log2FoldChange | and >=1 (L_JN vs D_JN) and padj≤ 0.05 is conditional filtering difference expression gene.

Based on transcript expression quantity FPKM value, by building FPKM value box traction substation and density map, on the whole to different rouge Transcript expression quantity in fat tissue samples is analyzed.The expression quantity point of the transcript of two breeding pig intramuscular fat tissues in group Cloth is than more consistent, and for transcript compared to Laiwu Pigs, low expression amount transcript is more in Large White adipose tissue between group.Turn between sample Record this expression analysis, it can be seen that experimental data meets the requirements on the whole.Simultaneously to the obtained lncRNA's of identification and mRNA Expression quantity is analyzed, and discovery mRNA has relatively high expression, and the expression quantity of lncRNA is lower, and FPKM value is mainly concentrated (0-10] between, FPKM value (0-100] between mRNA show and be uniformly distributed.

By the way that intramuscular fat tissue, ((L_JN vs D_JN) gene carries out Differential expression analysis (Fig. 1), and identification obtains altogether 56 differential expression lncRNAs (34 up-regulations, 22 downwards), (371 are raised 715 differential expression mRNAs, under 344 Adjust), wherein the gene with 4 times or more differences accounts for about 48.4%.Wherein using XLOC_007365 as the differential expression long-chain of representative Non-coding RNA and the research that us are included in using PLEKHG3, SERPINB7, SPTLC3, TMOD2 as the difference expression gene of representative Object.

9 difference expression gene GO and KEGG Pathway of embodiment enrichment analysis

Gene Ontology (Gene Ontology, GO, http://www.geneontology.org/) is gene function state Border classification standard, by molecular function (molecular function), biological process (biological process) and thin Born of the same parents' component (cellular component) composition.Access enrichment analysis can determine that the main metabolic way that difference expression gene participates in Diameter and signal path, KEGG (Kyoto Encyclopedia of Genes and Genomes, http: // Www.genome.jp/kegg) database (Kanehisa et al., 2008) is used as relevant main public database, be into The main tool that row metabolic analysis, regulated and control network are studied.In order to further study the principal biological function of difference expression gene, This experimental applications CluGO (Bindea et al., 2009) software, it is aobvious based on hypergeometric distribution checking computation difference expression gene The GO entry and signal path, the P value (Q_value) that Benjamini-Hochberg algorithm corrects for writing enrichment work as Q_ When value≤0.05, then enrichment is significant.

The difference expression gene that 513 databases have annotated is identified altogether in Large White and Laiwu Pigs intramuscular fat tissue, There are one or more entries of 210,144,62 genetic enrichments to biological process, molecular function and cellular component respectively, In largely with lipid-metabolism and the closely related GO entry significant enrichment of deposition.According to bioprocess, more gene (>=15) It is enriched to lipids, biological synthesis process (lipid biosynthetic process), Regulating Lipid Metabolism (lipid Metabolic process), cytolipin metabolic process (cellular lipid metabolic process), lipid answer Answer reaction (response to lipid), MAPK cascade reaction (MAPK cascade), MAPK cascade reaction just regulate and control (positive regulation of MAPK cascade) and MAPK cascade reaction regulate and control (regulation of MAPK cascade).For molecular function part, only significant enrichment is in inhibitor activity (enzyme inhibitor activity) Entry.In cellular component, significant enrichment is in the correlation such as extracellular matrix (extracellular matrix), aixs cylinder (axon) GO In entry.There are significant differences for Large White and Laiwu porcine intramuscular fat deposition, are annotated and are found by GO, difference expression gene is significant It is enriched in the bioprocess with lipid-metabolism and cell differentiation, shows the two intramuscular fat deposition, the molecular mechanism of metabolism exists Difference is regulated and controled by different genes.

The analysis of 10 difference expression gene protein-protein interaction network of embodiment

Interactions between protein research can disclose the function of protein from molecular level.Therefore, based on STRING (http: // String-db.org/) the interaction in protein interaction database carries out interactions between protein network point to difference expression gene Analysis, further to probe into phase interaction complicated between Large White and Laiwu Pigs intramuscular fat histological difference expressing gene coding albumen With relationship.Inclusion boar (Sus scrofa) in STRING database directly extracts differential gene collection column from database The interaction of table, can to obtained differential gene coding interactions between protein network data file progress using Cytoscape software It is analyzed depending on changing.In interactions between protein network, node (Node) is protein, and the interaction of edge (Edge) between albumen is closed System, degree (Degree) represent the protein amounts with specific node interaction, and node size is directly proportional to the degree of this node, section The color of point indicates the log2FoldChange value of difference expression gene.

The microRNA target prediction of 11 differential expression lncRNA of embodiment

As a kind of non-coding RNA, function is mainly reflected in the regulation to target gene lncRNA, mainly include to away from Trans acting regulatory (trans-regulation) from protein coding gene farther out, meanwhile, the base with identical expression pattern Cause functionally has strong correlation.Therefore by analyzing and researching lncRNA's to lncRNA and mRNA coexpression, trans Target gene.

By the Pearson correlation coefficients (Pearson for calculating differential expression lncRNA and mrna expression amount Correlation coefficient, PCC), the coexpression relationship of lncRNA and mRNA is analyzed, with | PCC | > 0.8 and P_ Value < 0.05 is the lncRNA-mRNA of threshold value screening coexpression.

LncRNAtrans acts on target gene analysis, by the interaction relationship between lncRNA and mRNA sequence, to difference The trans effect target gene of expression lncRNA is predicted that RNAplex (Tafer et al., 2011) software is for calculating Conjugated free energy (Energy) between lncRNA and mRNA sequence, in conjunction with coexpression as a result, with Energy<-20 and | PCC |>= 0.9 identification lncRNA trans acts on target gene.

By analysis search out XLOC_007365 trans related to fat metabolism act on target gene be PLEKHG3, SERPINB7, SPTLC3 and TMOD2 are higher in Laiwu Pigs intramuscular fat content relative to both Large Whites.

The quantitative fluorescent PCR of 12 differential expression lncRNA of embodiment is verified

This research is randomly selected in 9 difference tables of L_JN (Laiwu Pigs intramuscular tissue) vs D_JN (Large White intramuscular tissue) Up to gene (lncRNA 4, mRNA 5), each gene is arranged 3 biology and repeats, with pig actin β (actin Beta, ACTB) gene be internal reference, using the expression of qRT-PCR method validation gene.UsingPCR System 9700 (Applied Biosystems, USA) takes the RNA sample reverse transcription of about 0.5 μ g to synthesize cDNA template.It utilizesGreen PCR Kit (Qiagen, Germany) and480ⅡReal-time PCR Instrument (Roche, Swiss) carries out qRT-PCR analysis.

It will using HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme, R223-01) RNA reverse transcription to be measured is at cDNA.

(1) it takes out and deposits in the total serum IgE sample extracted under -80 DEG C of refrigerators, thaw at RT is as follows in 0.2mL PCR pipe Configure reverse transcription system.

(2) reverse transcription system (10 μ L): total serum IgE, 0.5 μ g；4 × gDNA wiper Mix, 2 μ L；Nuclease-free H₂O adds to 8 μ L, reaction condition: 42 DEG C of 2min.5 × HiScript II Q RT SuperMix IIa, 2 μ L are added, react item Part: 25 DEG C of 10min, 50 DEG C of 30min, 85 DEG C of 5min.

(3) Nuclease-free H is added after reverse transcription₂O is diluted to 100 μ L, -20 DEG C of preservations.

Real-time RCR reaction

(1) system configurations

Component and volume in 3 PCR system of table

(2) cycling condition

4 PCR cycle condition of table

3) PCR system is uniformly mixed, is centrifuged after reaction, assign to 384 orifice plates, In480ⅡReal- QRT-PCR reaction and analysis are carried out on time PCR Instrument (Roche, Swiss).

2- △ △ Ct method calculates the relative expression quantity of gene between each group sample, and t-, which is examined, carries out statistical to relative expression quantity Analysis, data are expressed as average ± standard deviation (Mean ± SD), and P < 0.05 indicates significant difference

FASN, XLOC_002561, XLOC_053194, CD36, MAP3K4 are significantly raised in Large White intramuscular fat, XLOC_027632, SCD significant up-regulated expression (Fig. 2) in Laiwu Pigs intramuscular fat.Result above is consistent with sequencing result, table Bright sequencing result is reliable.

It collects 10 Large White intramuscular tissue samples and 10 Laiwu Pigs intramuscular tissue samples carries out alternative gene XLOC_ 007365 and PLEKHG3 (XM_005666312.3), SERPINB7 (XM_001925047.5), SPTLC3 (XM_ 003134257.4), TMOD2 (XM_001925676.5) gene by fluorescence quantitative is verified, and specific steps are same as above.

Design of primers:

XLOC_007365:

Upstream primer: 5 '-AGGATGAAGTGAAACAATG-3 ' (SEQ ID NO.2)

Downstream primer: 5 '-GCGGATGCTCATATTTAC-3 ' (SEQ ID NO.3)

PLEKHG3 gene:

Upstream primer: 5 '-TACTCCTGTGGGTTGTCT-3 ' (SEQ ID NO.4)

Downstream primer: 5 '-TCAGCAGTGGTTATTACA-3 ' (SEQ ID NO.5)

SERPINB7 gene:

Upstream primer: 5 '-TTGCTCTTATCAGTTCTAC-3 ' (SEQ ID NO.6)

Downstream primer: 5 '-TGCTTCTCCATTACTATTC-3 ' (SEQ ID NO.7)

SPTLC3 gene:

Upstream primer: 5 '-ATTACATTCTCAACTCAAC-3 ' (SEQ ID NO.8)

Downstream primer: 5 '-CCTCTTAACCAACTTCAT-3 ' (SEQ ID NO.9)

TMOD2 gene:

Upstream primer: 5 '-TGTTCCTCTTCAGCAGTT-3 ' (SEQ ID NO.10)

Downstream primer: 5 '-CTCCTCTCCTTCACCAAT-3 ' (SEQ ID NO.11)

As a result see that Fig. 3 and Fig. 4, XLOC_007365 show high expression in Laiwu Pigs intramuscular fat, be the intramuscular rouge of Large White Nearly 4.8 times of fat tissue, PLEKHG3, SERPINB7, SPTLC3, TMOD2 gene are high table in Laiwu Pigs intramuscular fat It reaches, is 2.4 times, 13.5 times, 23.4 times and 2.2 times of Large White intramuscular fat tissue or so respectively.

The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.

Sequence table

<110>Institute of Animal Sciences, Chinese Academy of Agricultural Sciences

The new lncRNA of<120>one boar intramuscular fat tissue extractions and its application

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1327

<212> DNA

<213> Sus scrofa

<400> 1

gggtgggggt gctgaccagt ggccccacca gctcttaagc agctgctgcc tctgtgtgcc 60

aggctcaggg gttcgctcag aggggtgagg tggtgggaag ggggtgacct gtgcagggca 120

ggtgctctga aggacgggga cagggacctc ccccaggtgg gagcctgggc ccaggagggg 180

agcttgcttc ctgcttctcc ccattgatgc atgaagaagg tggctcccag ggagctgagg 240

cagctttcct gggggcacag ccgcgagggg aggggctggg ccttgaacct gcctcaggct 300

ccccgtgccc atgggcttgc ccttggcgtc cctgggccta taattcagca gggagggctt 360

cctggaggga tggtccttgt tggaggatgc agaggaggag ggagcagtga aaccttcccc 420

aggggggctg cggggcccct ggggacctgc ggggtttgtg cagccggaca tgaggtgggc 480

tccctttgtc aggagaggac ggagggcccc tcctgagctt ctcctggcat cacggctgcc 540

aggcacagcc cacaccccag ggacttaccc cctccgccca ccgggagggg tggccctaac 600

gctcagtgac aggtgtgcag ctggcgctca gtgacaagac atctttaacc agaccctggc 660

cgtggccttt cccacgtccc gggaagagac atcgatctga gtctaaaagg agcagaaggg 720

acagcagttc ctggaccgat ggggacatcg tcccagggac aggatgaagt gaaacaatga 780

ctcaggaccc aaatgccaca cttggctggg aaacagcttc cataggagta aatatgagca 840

tccgccatgc acaccaagca gggacctggg ccgggagcgc gttttcccgg cggtgcgtgt 900

ttccctgaaa cgctggcttt gaagacttcg ggtggggaat agtcctccct tgaagcgcat 960

ctgtcctggg tgagaacccc cctcccgggc ccccgtctcc ccttttgcaa agcgggggct 1020

ccctgagctc tcacgtgggg acctcgggga aggcaggcca ccgtggacct caatgtcccc 1080

cgcccgcctt catcactggg agaaacgcaa gccagatgtg aagcagcaaa cgccctcagt 1140

ctgggcgtgg acccacccca agtcctctag acccggtggg tcgtggtgga cggtcctggc 1200

cctggcgctg cggctccggg acttgggggt ttgaagagaa gtgcggctcc gtggagacgt 1260

ggcaagaccc gtaccgcctg gtgctgggtt cctgcccttc ccggaccccc ggctggaccg 1320

cgtgccc 1327

<210> 2

<211> 19

<212> DNA

<213> Sus scrofa

<400> 2

aggatgaagt gaaacaatg 19

<210> 3

<211> 18

<212> DNA

<213> Sus scrofa

<400> 3

gcggatgctc atatttac 18

<210> 4

<211> 18

<212> DNA

<213> Sus scrofa

<400> 4

tactcctgtg ggttgtct 18

<210> 5

<211> 18

<212> DNA

<213> Sus scrofa

<400> 5

tcagcagtgg ttattaca 18

<210> 6

<211> 19

<212> DNA

<213> Sus scrofa

<400> 6

ttgctcttat cagttctac 19

<210> 7

<211> 19

<212> DNA

<213> Sus scrofa

<400> 7

tgcttctcca ttactattc 19

<210> 8

<211> 19

<212> DNA

<213> Sus scrofa

<400> 8

attacattct caactcaac 19

<210> 9

<211> 18

<212> DNA

<213> Sus scrofa

<400> 9

cctcttaacc aacttcat 18

<210> 10

<211> 18

<212> DNA

<213> Sus scrofa

<400> 10

tgttcctctt cagcagtt 18

<210> 11

<211> 18

<212> DNA

<213> Sus scrofa

<400> 11

ctcctctcct tcaccaat 18

Claims

1. a kind of lncRNA relevant to pig intramuscular fat, which is characterized in that lncRNA sequence is SEQ ID NO.1.

2. the application of lncRNA described in claim 1, which is characterized in that the lncRNA is for predicting or assisting prediction pig Application in meat；Or the lncRNA is used to prepare the application in prediction or auxiliary prediction meat quality reagent；Or institute The lncRNA stated has the application in different meat quality pigs for breeding.

3. a kind of reagent for detecting pig intramuscular fat, which is characterized in that reagent detects claim 1 institute by nucleic acid amplification technologies The expression of the lncRNA stated, the primer sequence for nucleic acid amplification are SEQ ID NO.2 and SEQ ID NO.3.

4. reagent according to claim 3, which is characterized in that the sample of reagent detection is tissue.

5. reagent according to claim 3, which is characterized in that the sample of reagent detection is intramuscular fat tissue.

6. the application of reagent described in claim 3-5 any one, which is characterized in that the reagent is for predicting or assisting Predict the application in meat quality；Or the reagent is used to prepare the application in prediction or auxiliary prediction meat quality reagent； Or the reagent has the application in different meat quality boars for breeding.

7. a kind of reagent for detecting pig intramuscular fat, reagent detects the lncRNA's in claim 1 by nucleic acid amplification technologies The expression of target gene, target gene are PLEKHG3 and/or SERPINB7 and/or SPTLC3 and/or TMOD2, and feature exists In reagent includes a pair of for expanding the primer of the target gene of lncRNA, and primer sequence is selected from: by SEQ ID NO.4 and SEQ The primer pair of ID NO.5 composition；The primer pair being made of SEQ ID NO.6 and SEQ ID NO.7；By SEQ ID NO.8 and SEQ The primer pair of ID NO.9 composition；The primer pair being made of SEQ ID NO.10 and SEQ ID NO.11.

8. reagent as claimed in claim 7 is characterized in that using the phase, the reagent is for predicting or assisting prediction pork product Application in matter；Or the reagent is used to prepare the application in prediction or auxiliary prediction meat quality reagent；Or the examination Agent has the application in different meat quality pigs for breeding.