CN108330170B - A kind of application of lncRNA in the detection of tibetan sheep hypoxia adaptability - Google Patents

Abstract

The present invention provides application of the lncRNA in the detection of tibetan sheep hypoxia adaptability, the lncRNA is TCONS_00332125, TCONS_00377466 or TCONS_00139593, pass through system research tibetan sheep hypoxia adaptability lncRNA express spectra, screen the lncRNA relevant to hypoxia adaptability verified through qRT-PCR, it can be applied to the detection of tibetan sheep hypoxia adaptability, help to illustrate tibetan sheep to the Adaptive mechanism under low-oxygen environment stress, basic theory in terms of abundant extreme environment ruminant molecular ecology adaptability, and scientific support is provided for tibetan sheep molecular breeding and functional genomics research.

Description

A kind of application of lncRNA in the detection of tibetan sheep hypoxia adaptability

Technical field

The present invention relates to molecular biology fields, and in particular to a kind of lncRNA is in the detection of tibetan sheep hypoxia adaptability Using.

Background technique

Qinghai-Tibet tibetan sheep is broadly divided into plateau type (Grassland Type), mountain valley-type and Euler's type three categories, and various regions are according to it Ecological characteristic is subdivided into different types, belongs to coarse wool type sheep local varieties.Tibetan sheep originates in Qinghai-Tibet Platean, mainly The ground such as Tibet Autonomous Region and Qinghai, Gansu, Sichuan, Yunnan, Guizhou are distributed in, due to each ecological condition most diverse, are formd Different types.Plateau type (Grassland Type) tibetan sheep is main body, and quantity is most, be distributed mainly on the domestic Gangdise in Tibet, Northern Tibet Plateau and Yarlung Zangbo River area to the north of Nyainqentanglha Shan；Tibetan sheep is mainly distributed on Southern Qinghai Province Banma, two county of Nangqian some areas, Sichuan Province Abazangzuqiangzu Autonomous Prefecture south pastoral area is Zhaotong County, Yunnan city, Qujing, beautiful Jiang Shi and Baoshan City Tengchong County.Euler's type tibetan sheep is a special ecological type, and center producing region is located at Gansu Province's Gannantibetan Maqu County Euler township of autonomous prefecture and neighbour area and the ground such as Qinghai Province Mongolian Autonomous County of Henan and Jiuzhi County.

High and cold and anoxic is the main ecological restriction factor in highlands, and plateau original inhabitants animal is in long-term adaptive evolution Formd in journey unique Hypoxia adaptation strategy (Liu et al., 2016；Wei et al.,2015；Beall,2013).Hiding is continuous Sheep be Qinghai-Tibet Platean indigenous ruminant domestic animal and Grassland ecosystems important component, be the important life of Tibetan people and The means of production, while being also the symbol (Long et al., 2008) of its wealth.Tibetan sheep is in extensive and traditional grazing management Under mode, ecological environment harsh to Qinghai-Tibet Platean over the past thousands of years has extremely strong adaptability.Tibetan sheep passes through for a long time oneself So selection and evolution have the severe natural environmental conditions such as high and cold, hypoxemia, intensive ultraviolet and cold season Nutrient Stress extremely strong Adaptability, and adjusted by physiology and Nutrition and Metabolism.In the lung group to High aititude tibetan sheep and low altitude area Small-fat-tail sheep Knit in structural research, discovery tibetan sheep unit area alveolar number it is more significant than Small-fat-tail sheep increase, the thickness of alveolar septa dramatically increases, The caliber of bronchioli terminales is substantially reduced, and think these differences and feature be tibetan sheep lung tissue adapt to Alpine cold and hypoxia shape State feature (Yu Hongxian, 1999).Measure pulmonary arterial pressure, pulmonary arterial wedge pressure, body arterial pressure and cardiac output, the calculating of tibetan sheep Pulmonary vascular resistance, under the action of NOS non-selective inhibitor, tibetan sheep pulmonary arterial pressure is had increased slightly, and cardiac output is reduced, and 4500 meters than 2300 meters at the pulmonary vascular resistance of tibetan sheep significantly increase (P < 0.05)；Under the effect of NOS selective depression object, only There is the former pulmonary vascular resistance to increase, the latter does not change.It being suppressed with endogenous NO S activity, the production quantity of NO is also reduced, Promote body Pulmonary Vascular to shrink, tibetan sheep is made to adapt to low-oxygen environment (Ruan et al., 2004).This and Koizumi etc. are to difference The measurement result of the pulmonary vascular resistance of height above sea level tibetan sheep is consistent (Koizumi et al., 2004).Under the conditions of chronic hypoxia, draw Expression quantity into VEGF the and NOS gene in High aititude sheep placenta raises, and body alleviates hypoxemia by synthesis VEGF, this can Can be low altitude area sheep high altitude localities long-term acclimatization mechanism (Parraguez et al., 2010).It is hidden to High aititude In sheep and the research of low altitude area sheep biochemical indicator, tibetan sheep biochemical indicator (red blood cell, hematocrit, mean corpuscular Volume, mean corpuscular hemoglobin) it is high or significantly high, tibetan sheep is not from increase hemoglobin to the adaptation of Alpine cold and hypoxia It is realized in concentration, it may be possible to improve the ability and efficiency of hemoglobin conveying oxygen, these unique physiological functions have Conducive to increase pulmonary ventilation volume and pulmonary blood flow volume, and it is high and cold low to adapt to adjust pulmonary blood vasculature by endogenous NO yield height The environment (Liu et al., 2016) of oxygen.

Tibetan sheep is not simply formed with effect intake, conveying and structure feature and physiological mechanism using oxygen, and by thin Born of the same parents' metabolism and the induction of the anti-hypoxemia factor adapted to from molecular level Alpine cold and hypoxia environment (Wei et al., 2016；Yang et al.,2016；Liu et al.,2016).This is mainly manifested in some cell factors, lymphokine, hormone, energetic supersession, oxygen In the variation of some adaptability related genes such as transmission, response anoxic, DNA repair enzyme and signal path；In low-oxygen environment Histocyte then may experience hypoxia signal by the oxygen acceptor molecule on its film, and start many hypoxic effect genes and do Complicated response (Wu, 2012) out.

In recent years with the innovation of technique of gene detection, high and cold original inhabitants animal adapt to the Mysterious Veil of Alpine cold and hypoxia environment by by Step is opened.By carrying out genome-wide screening to the individual of native sheep breeds 122 of Different Altitude gradient 7, adaptation is disclosed 236 differential genes and 8 specific candidate genes of Alpine cold and hypoxia environment, wherein EPAS1 gene regulates and controls net in High aititude hypoxemia Central role is played in network, at the same also contain three Gene A dam17, Arg2 relevant to hypoxia inducible factor HIF-1 α, Mmp3(Wei et al.,2016；Liu et al.,2016).Using high throughput sequencing technologies and analysis of biological information strategy, from Genomic level sufficiently discloses the distinctive height of Tibetan pig (Li et al., 2013) and ground titmouse (Qu et al., 2013) Cold hypoxia adaptability molecular mechanism.In terms of feed digestion metabolism, tibetan sheep rumen microorganism group is improved by gene regulation Diet dry matter, crude protein, digestibility of fiber, nitrogen retention, daily ration nitrogen use efficiency, effective urea Transform efficiency (Huang andLiu,2009；Zhou Jianwei, 2015), disclose tibetan sheep nitrogen and coerce lower nutrition flexibility mechanism.

Tibetan sheep is distinctive, ancient, the best in quality carpet wool sheep local group in China Qinghai-Tibet Platean.Due to hiding Sheep can adapt to severe puna environment, make full use of other unserviceable natural resources of big domestic animal.Because it is long-term It lives in hypoxemia, cold, drying, radiate strong, little precipitation, the highlands that 4000 meters of mean sea level, in environmental pressure and slightly It puts under formula feeding manner, has formed the genetic mechanism stable to Alpine cold and hypoxia, dry habitat.

Long-chain non-coding RNA (longnon-coding RNA, lncRNA) is a kind of positioned at nucleus or intracytoplasmic Functional RNA molecule, by rna plymerase ii (RNAPolymerase II) or rna plymerase iii (RNAPolymerase III it) transcribes, repetitive sequence is less, and half-life period is shorter, and binding site is single, be a kind of transcript length is more than 200 The non-coding RNA of nucleotide itself does not encode albumen, but in the expression of a variety of level controlling genes in the form of RNA It is horizontal.Research in recent years discovery lncRNA is a kind of RNA with important biomolecule function, participates in transcriptional activation, transcription interference, core A variety of important regulation processes such as interior transport, chromosome modification, chromosome silencing, genomic imprinting, heredity and epigenetic, Important regulating and controlling effect is played in the vital movements such as genetic transcription and translation, cell differentiation and development.

But the Adaptive mechanism under the prior art coerces low-oxygen environment is not known, without any about lncRNA In the application of tibetan sheep hypoxia adaptability context of detection, tibetan sheep hypoxia adaptability correlation function is not carried out using lncRNA yet The method of energy genetic test.

Summary of the invention

In view of this, the application the purpose of the present invention is to provide lncRNA in the detection of tibetan sheep hypoxia adaptability.

In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:

The application that the present invention provides lncRNA in tibetan sheep liver and lung tissue in hypoxia adaptability detection, it is described LncRNA is TCONS_00332125, TCONS_00377466 or TCONS_00139593.

The screening technique of the lncRNA the following steps are included:

1) extracting has the duplicate Different Altitude tibetan sheep of 3 biology and control group sheep liver and lung tissue sample LncRNA sequencing is carried out, Clean Data data are obtained by analysis；

2) lncRNA that the Clean Data data obtain prediction is analyzed；

3) significant difference in Different Altitude tibetan sheep and control group sheep liver and lung tissue sample is respectively compared to express Candidate lncRNA be detected hypoxia adaptability lncRNA.

Preferably, the Different Altitude tibetan sheep includes High aititude bar tibetan sheep and A Wang tibetan sheep and intermediate altitude suddenly The white tibetan sheep in Qilian and Gan Jia tibetan sheep.

Preferably, the step 1) the following steps are included:

1.1) total serum IgE of tibetan sheep and sheep liver and lung tissue sample is extracted；

1.2) cDNA library that the step 1.1) extracts total serum IgE is constructed；

1.3) cDNA library of the building of step 1.2) described in quality inspection；

1.4) machine on the cDNA library of the total serum IgE is sequenced, Clean Data data are obtained by analysis.

Preferably, the step 2) the following steps are included:

2.1) the Clean Data data are pre-processed and is uniformed, so that Q30 base percentage is greater than 85%, obtain Obtain valid data；

2.2) valid data and reference genome Oar_v3.1 obtained the step 2.1) carry out sequence alignment, obtain Mapped Reads；

2.3) the Mapped Reads is subjected to transcript splicing using Cufflinks and Scripture program；

2.4) transcript for selecting length >=200bp, Exon number >=2, calculates each transcript using cufflinks Minimum vertex-covering degree, the transcript for selecting minimum vertex-covering degree >=3, or spliced simultaneously by Cufflinks and Scripture LncRNA is the lncRNA of prediction.

The present invention also provides a kind of kits of hypoxia adaptability in detection tibetan sheep liver and lung tissue, including with Lower component: the reverse transcription system of total serum IgE and the amplification system of cDNA；It include total serum IgE in the reverse transcription system of the total serum IgE, it is inverse Transcriptase, dNTP and oligdT；It include TCONS_00332125 primer, TCONS_00377466 in the amplification system of the cDNA Primer and TCONS_00139593 primer, the TCONS_00332125 primer have SEQ ID NO.4 and SEQ ID NO.5 institute Show that nucleotide sequence, the TCONS_00377466 primer have nucleotides sequence shown in SEQ ID NO.6 and SEQ ID NO.7 Column, the TCONS_00139593 primer have nucleotide sequence shown in SEQ ID NO.8 and SEQ IDNO.9.

The present invention also provides the application methods of the kit, comprising the following steps: it is total to extract test serum sample RNA；The total serum IgE reverse transcription of extraction is prepared into cDNA using the total serum IgE reverse transcription system；Body is expanded using the cDNA System, is prepared with the TCONS_00332125 primer, TCONS_00377466 primer and TCONS_00139593 primer pair CDNA carry out qRT-PCR expand lncRNA.

The application that the present invention provides lncRNA in tibetan sheep liver and lung tissue in hypoxia adaptability detection, it is described LncRNA is TCONS_00332125, TCONS_00377466 or TCONS_00139593, passes through cis the and trans mode of action Predict 10 target genes of the lncRNA.

Preferably, the qRT-PCR amplimer of the hypoxia adaptability lncRNA target gene includes SEQ ID NO.10- 29。

Beneficial effects of the present invention

LncRNA of the present invention includes TCONS_00332125, TCONS_00377466 or TCONS_00139593, It can be applied to the detection of tibetan sheep hypoxia adaptability, help to illustrate the Adaptive mechanism under tibetan sheep coerces low-oxygen environment, Basic theory in terms of abundant extreme environment ruminant molecular ecology adaptability, and be tibetan sheep molecular breeding and function base Because group research provides science support.

Detailed description of the invention

Fig. 1 is the tibetan sheep differential expression lncRNA screened in liver organization；

Fig. 2 is the tibetan sheep differential expression lncRNA screened in lung tissue；

Fig. 3 is the tibetan sheep hypoxia adaptability lncRNA screened in liver organization；

Fig. 4 is the tibetan sheep hypoxia adaptability lncRNA screened in lung tissue；

Fig. 5 is the qRT-PCR verification result of liver and lung tissue tibetan sheep hypoxia adaptability lncRNA and its target gene.

Specific embodiment

It is described the present invention provides application of the lncRNA in tibetan sheep liver and the detection of lung tissue hypoxia adaptability LncRNA is TCONS_00332125, TCONS_00377466 or TCONS_00139593.

In the present invention, the screening technique of the lncRNA preferably includes following steps:

1) Different Altitude tibetan sheep and control group sheep liver are extracted and lung tissue sample carries out lncRNA sequencing, is passed through Analysis obtains Clean Data；

2) analysis Clean Data data obtain the lncRNA of prediction；

3) compare the time that significant difference is expressed in Different Altitude tibetan sheep and control group sheep liver and lung tissue sample Selecting lncRNA is detected hypoxia adaptability lncRNA.

The Different Altitude tibetan sheep specifically includes High aititude bar tibetan sheep and A Wang tibetan sheep suddenly in the present invention；Institute State that a bar tibetan sheep height above sea level suddenly is 4468m, the A Wang tibetan sheep height above sea level is 4452m；The white tibetan sheep in intermediate altitude Qilian With sweet plus tibetan sheep；The white tibetan sheep height above sea level in Qilian is 3520m, and described sweet plus tibetan sheep height above sea level is 3551m；This Control group sheep height above sea level is -67m in invention.The TCONS_00332125 primer has SEQ ID NO.4 and SEQ ID Nucleotide sequence shown in NO.5, the TCONS_00377466 primer have nucleosides shown in SEQ ID NO.6 and SEQ ID NO.7 Acid sequence, the TCONS_00139593 primer have nucleotide sequence shown in SEQ ID NO.8 and SEQ ID NO.9.

The present invention preferably acquires the liver and lung tissue of above-mentioned each tibetan sheep and sheep sample from different places, point Indescribably take liver and lung tissue total serum IgE.The extraction of heretofore described liver and lung tissue total serum IgE preferably uses Life Technologies companyReagent carries out, and specific operating procedure is shown inReagent article No. is 15596026 specification.

The present invention preferably uses 2000 spectrophotometer of NanoDrop after extracting acquisition liver and lungs total serum IgE (Thermo Scientific, USA) measures extracted total serum IgE purity and concentration, determines the OD of total serum IgE₂₆₀/OD₂₈₀In 1.8- Between 2.1, the concentration of total serum IgE is more than or equal to 100ng/ μ L.The present invention is determining that it is above-mentioned that the total serum IgE purity extracted and concentration meet After condition, then the total serum IgE integrality of extraction is detected, the detection of the total serum IgE integrality is preferably solidifying using agarose There are clear two bands (28S/18S rRNA), have third strip (5S sometimes in the method for gel electrophoresis quality inspection, electrophoresis RRNA), the top not may occur in which that DNA pollution band, 28S/18S are answered twice approximate.

The present invention is after the purity, concentration and integrality for determining liver and lung tissue total serum IgE all meet the requirements, preferred structure Build the cDNA library of RNA, the construction cDNA library it is specific the following steps are included:

1.21) using the rRNA in epicentre Ribo-ZeroTM kit removal sample total serum IgE, rRNA- is obtained DepletedRNA, specific steps are operated according to epicentre Ribo-ZeroTM kit specification；

1.22) Fragmentation Buffer is then added into the rRNA-depleted RNA, by rRNA- Depleted RNA is interrupted at random；

1.23) using the rRNA-depleted RNA interrupted immediately as template, with hexabasic base random primer (randomhexamers) synthesize first cDNA chain, then into first cDNA chain be added buffer, dATP, DUTP, dCTP, dGTP, RNase H and DNApolymerase I synthesize Article 2 cDNA chain, utilize AMPure XPbeads couple The double stranded cDNA purification of synthesis；

1.24) end reparation plus A are successively carried out to the double-strand cDNA of the purifying and connects sequence measuring joints, use AMPure The segment of XPbeads selection 180-200bp size；

1.25) the double-strand cDNA degradation chain containing U obtained the step 1.24), is enriched with to obtain cDNA library by PCR.

The present invention preferably carries out library Quality Control, library matter to the cDNA library of acquisition after the cDNA library for obtaining RNA Machine sequencing is carried out after control is qualified, Clean Data is obtained by analysis.The library Quality Control preferably uses Qubit 2.0 It is detected with concentration and Insert Fragment size (Insert Size) of the Agilent 2100 to library, Insert Fragment size is excellent Choosing is 180-200bp；Accurate quantitative analysis is carried out using effective concentration of the Q-PCR method to library, the effective concentration in library is greater than 2nM is qualification.

The present invention carries out machine sequencing, the sequencing preferably carries out high pass with HiSeq2500 after library Quality Control is qualified Sequence, sequencing reading length PE125 are measured, each library sample sequencing output is no less than 10Gb CleanData.

The present invention analyzes Clean Data data, obtains prediction after obtaining Clean Data data lncRNA.In the present invention analyze Clean Data data the following steps are included:

2.1) Clean Data data are pre-processed and is uniformed, so that Q30 base percentage is greater than 85%, had Imitate data；

2.2) valid data and reference genome Oar_v3.1 obtained step 2.1) carry out sequence alignment, obtain Mapped Reads；

2.3) Mapped Reads is subjected to transcript splicing using Cufflinks and Scripture program；

2.4) transcript for selecting length >=200bp, Exon number >=2, calculates each transcript using cufflinks Minimum vertex-covering degree, the transcript for selecting minimum vertex-covering degree >=3, or spliced simultaneously by Cufflinks and Scripture LncRNA is the lncRNA of prediction.

The present invention detects differential expression lncRNA, in differential expression lncRNA detection process after obtaining prediction lncRNA In, by Change>=2 Fold and FDR<0.01 is used as screening criteria.After the present invention filters out differential expression lncRNA, compare respectively It is to detect compared with the candidate lncRNA that significant difference in Different Altitude tibetan sheep and control group sheep liver and lung tissue is expressed The hypoxia adaptability lncRNA of acquisition.

In the present invention, the qRT-PCR primer of the lncRNA is to be designed according to lncRNA sequence with primer-design software Synthesis, TCONS_00332125 upstream and downstream primer is respectively SEQ ID NO.4 and SEQ in the preferred lncRNA ID NO.5, TCONS_00377466 upstream and downstream primer are respectively SEQ ID NO.6 and SEQ ID NO.7, TCONS_ 00139593 upstream and downstream primer is respectively SEQ ID NO.8 and SEQ ID NO.9.

Present invention extraction liver described during verifying detects hypoxia adaptability lncRNA and lung tissue sample are total RNA step extracts liver with the screening technique of above-mentioned lncRNA and lung tissue sample total serum IgE step is consistent, no longer superfluous herein It states.

The present invention carries out the preparation of tissue sample cDNA after extracting tissue sample total serum IgE.Heretofore described tissue sample Preparing for product cDNA is preferred are as follows: carries out reverse transcription synthetic tissue sample cDNA to the tissue sample total serum IgE.Specifically in this hair It is bright middle using Vazyme kit HiScript II Q RT SuperMix for qPCR (+gDNAwiper) (article No. R223- 01) reverse transcription is carried out to the tissue sample total serum IgE of extraction and synthesizes cDNA.Specific reaction includes two steps: the first step removes base Because group DNA and second step reverse transcription synthesize cDNA.First step removal genomic DNA reaction, reaction system such as table 1 in the present invention

The reaction system of the removal genomic DNA of table 1

Reagent	Usage amount
		4×gDNAwiperMix	2.0μL
TotalRNA	0.5μg
		Nuclease-freeH2O	Add to 8.0 μ L

Mentioned reagent needed for the present invention reacts the first step mixes and is placed on 42 DEG C of reaction 2min.

Reaction system such as table 2 by second step reverse transcription synthesis cDNA in the present invention

The reaction system of 2 reverse transcription of table synthesis cDNA

Reagent	Usage amount
		The reaction solution of the first step	8.0μL
5×HiScriptIIQRTSuperMixII	2.0μL
		Total volume	10.0μL

The present invention exists above-mentioned second step reaction component after mixing480 II type quantitative fluorescent PCRs It is reacted on instrument (Roche, Swiss), response procedures are as follows: 5min at 30min → 85 DEG C at 10min → 50 DEG C at 25 DEG C.It reverses After record, 90 μ L Nuclease-free H are added into obtained reverse transcription system by the present invention₂O is diluted to 100 μ L, obtains cDNA.In the present invention, the cDNA is spare under the conditions of being stored in -20 DEG C.

The present invention is after obtaining cDNA, using the cDNA as template, is expanded with the qRT-PCR primer pair cDNA of lncRNA Increase.It is specific in the present invention to useGreen PCRKit kit (Qiagen, Germany) QRT-PCR amplification is carried out, is existed using the cDNA of above-mentioned acquisition as template480 II type fluorescence quantitative PCR instruments Fluorescent quantitative PCR is carried out on (Roche, Swiss).The fluorescent quantitative PCR system is as shown in table 3.

3 fluorescent quantitative PCR system of table

Quantitative fluorescent PCR program: 95 DEG C of 5min；95 DEG C of 10s, 60 DEG C of 30s, 40 circulations；Melting is utilized after circulation terminates Curve detection product specificities are to slowly warm up to 97 DEG C from 60 DEG C, 5 fluorescence signals of every 1 DEG C of acquisition.

The present invention also provides application of the tibetan sheep hypoxia adaptability lncRNA in the detection of its target gene, the lncRNA For TCONS_00332125, TCONS_00377466 or TCONS_00139593, the tibetan sheep hypoxia adaptability lncRNA leads to It crosses cis the and trans mode of action and its target gene interacts.The Cis mode of action is that a kind of target gene of lncRNA is made With mode, the target gene of the hypoxia adaptability lncRNA preferably within the scope of lncRNA upstream and downstream 10kb, is preferably opened The target gene that sub-area is transcribed in the same direction is usually to promote expressional function, is reversed inhibition.And in 3 '-UTR region, part feelings Reversed under condition is also promotion expression.

The expression quantity of heretofore described hypoxia adaptability lncRNA and its target gene is tested by qRT-PCR amplification Card.The method and steps of the expression quantity qRT-PCR amplification of heretofore described hypoxia adaptability lncRNA is with above-mentioned lncRNA's QRT-PCR detection method is consistent, and details are not described herein.The qRT-PCR amplification of the hypoxia adaptability lncRNA target gene is drawn Object preferably includes SEQ ID NO.10-29.

By to detected hypoxia adaptability differential expression lncRNA carry out functional annotation, enrichment (GO, KEGG) and Interactions between protein network analysis helps to illustrate tibetan sheep to the Adaptive mechanism under low-oxygen environment stress, enriches extreme environment Basic theory in terms of ruminant molecular ecology adaptability, and provided for tibetan sheep molecular breeding and functional genomics research Science support.

Present invention will be further explained below with reference to specific examples, for explaining only the invention, and should not be understood as to this The limitation of invention.It will be understood by those skilled in the art that: without departing from the principle and spirit of the present invention may be used To carry out a variety of change, modification, replacement and modification to these embodiments, the scope of the present invention is limited by claim and its equivalent It is fixed.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition or according to item proposed by manufacturer Part examinations.

Embodiment 1

One, material and method

1, material

Tibetan sheep liver and lung tissue sample information are as shown in table 4

4 tibetan sheep liver of table and lung tissue sample information

2, method

The extraction of 2.1 tibetan sheep livers and lung tissue total serum IgE

According to Life Technologies companyReagent (article No. 15596026) specification extract liver and The Total RNA of lungs, then extracted with 2000 spectrophotometer of NanoDrop (Thermo Scientific, USA) measurement RNA purity and concentration, agarose gel electrophoresis quality inspection ensure extract RNA integrality.

2.2RNA library construction

2.2.1 sample rRNA is removed using epicentre Ribo-ZeroTM kit；

2.2.2 FragmentationBuffer is added to interrupt rRNA-depleted RNA at random；

2.2.3 it using rRNA-depleted RNA as template, is synthesized with hexabasic base random primer (random hexamers) Then buffer, dATP, dUTP, dCTP, dGTP, RNase H and DNApolymerase I synthesis is added in first cDNA chain Article 2 cDNA chain purifies cDNA using AMPure XP beads；

2.2.4 the double-strand cDNA purified carries out end reparation plus A again and connects sequence measuring joints, then uses AMPure XPbeads carries out clip size selection；

2.2.5 last degradation chain containing U, is enriched with to obtain cDNA library by PCR.

2.3 library Quality Controls

2.3.1 carried out using Qubit 2.0 it is tentatively quantitative, using Agilent 2100 to the Insert Size in library into Capable detection, Insert Size can just carry out next step experiment after meeting expection；

2.3.2Q-PCR method carries out accurate quantitative analysis (library effective concentration > 2nM) to the effective concentration in library, completes library Inspection.

Machine is sequenced on 2.4

After library inspection is qualified, high-flux sequence, sequencing reading length PE125 are carried out with HiSeq2500, output is sequenced in each sample No less than 10Gb Clean Data.

3, data are analyzed

3.1 sequencing datas and its quality control

It is controlled by sequencing quality, obtains 379Gb CleanData altogether, Clean Data data obtained are counted Data preprocess and homogenization, each sample Q30 base percentage reach 85% or more.

3.2 long-chain non-coding data are compared with reference to genome sequence

Use genome Oar_v3.1 as with reference to progress sequence alignment and subsequent lncRNA analysis, Oar_v3.1 downloading ground Location://ftp.ensembl.org/pub/release-78/fasta/ovis_aries/。

The assessment of 3.3 long-chain non-coding Library Qualities

Qualified long-chain non-coding library is the necessary condition of long-chain non-coding sequencing, for the quality for ensuring library, from Lower 3 different angles carry out quality evaluation to long-chain non-coding sequencing library:

3.3.1 by examining distribution of the Mapped Reads on transcript, randomness, the mRNA of mRNA fragmentation are assessed Degradation situation；

3.3.2 by the distribution of lengths of Insert Fragment, the dispersion degree of Insert Fragment length is assessed；

3.3.3 it by drawing saturation degree figure, assesses library capacity and whether Mapped Data is sufficient.

The splicing of 3.4 transcripts

Mapped Reads is subjected to transcript splicing using Cufflinks and Scripture program.

3.5 lncRNA screening

LncRNA basic screening includes: the mRNA (transcript and its spliceosome) removed in genome database, selection length The transcript of degree >=200bp, Exon number >=2, the minimum vertex-covering degree for calculating each transcript using cufflinks, selection are most The transcript of small coverage >=3, while also being screened by the lncRNA that Cufflinks and Scripture splice, it obtains Final tibetan sheep hypoxia adaptability lncRNA detection.

4, result

4.1 the present invention using Cufflinks and Scripture assembling obtain 473766 transcripts, by CPC, PFAM, CNCI, CPAT encode potency analysis, and identification obtains 8107 lncRNA (Fig. 1) in liver organization, are combined by Different Altitude Comparative analysis shares 79 differential expression lncRNA；Identification obtains 1798 lncRNA (Fig. 2) in lung tissue, passes through different seas It pulls out combination comparative analysis and shares 151 differential expression lncRNA.

4.2 present invention relatively Different Altitude tibetan sheep and control group sheep liver organization share 2 Hypoxia adaptation specificity Candidate differential expression lcnRNA (Fig. 3), lung tissue share 1 Hypoxia adaptation specific candidate differential expression lcnRNA (Fig. 4).

4.3 present invention predict 10 of 3 tibetan sheep hypoxia adaptability lncRNA by cis the and trans mode of action Target gene.

Embodiment 2

1, material: with embodiment 1.

2, method

2.1 Hypoxia adaptation specific candidate differential expression lncRNA and its target gene design of primers: it is used for dye class qRT- The primer 13 of PCR verifying is right: primer sequence, annealing temperature and product length information are as shown in table 5 below.

5 primer sequence of table, annealing temperature and product length information

The extraction of 2.2 tibetan sheeps and sheep liver and lung tissue total serum IgE

With embodiment 1.

The preparation of 2.3 livers and lung tissue sample cDNA

Using Vazyme kit HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (article No. R223-01 cDNA) is synthesized to the total serum IgE reverse transcription of extraction.

The first step removes genomic DNA reaction, and reaction system and condition are as shown in table 6.

Table 6 removes genomic DNA reaction system and condition

Reagent	Usage amount
		4×gDNAwiperMix	2.0μL
TotalRNA	0.5μg
		Nuclease-freeH2O	Add to 8.0 μ L

42 DEG C of reaction 2min after above-mentioned component is mixed；

Second step reverse transcription reaction, reaction system and condition are as shown in table 7

7 reverse transcription reaction system of table and condition

Reagent	Usage amount
		The reaction solution of the first step	8.0μL
5×HiScriptIIQRTSuperMixII	2.0μL
		Total volume	10.0μL

Above-mentioned component is existed after mixing480 II type fluorescence quantitative PCR instruments (Roche, Swiss) Upper reaction, 25 DEG C of 10min, 50 DEG C of 30min, 85 DEG C of 5min；90 μ L Nuclease-free H are added after reverse transcription₂O is dilute It releases to 100 μ L, it is spare to be stored in -20 DEG C of refrigerators.

The expression quantity of 2.4 dye class fluorescence quantitative PCR detection specific candidate differential expression lncRNA and its target gene

UsingGreenPCR Kit kit (Qiagen, Germany), with reverse transcription CDNA is that template existsQuantitative fluorescent PCR expansion is carried out on 480 II type fluorescence quantitative PCR instruments (Roche, Swiss) Increase.

Dye class quantitative fluorescent PCR system is as shown in table 8.

8 dye class quantitative fluorescent PCR system of table

Quantitative fluorescent PCR program: 95 DEG C of 5min；95 DEG C of 10s, 60 DEG C of 30s, 40 circulations；Melting is utilized after circulation terminates Curve detection product specificities are to slowly warm up to 97 DEG C from 60 DEG C, 5 fluorescence signals of every DEG C of acquisition.

2.5 according to the relative quantification formula of qRT-PCR calculate Hypoxia adaptation specific candidate differential expression lncRNA and its The expression of target gene

According to the relative quantification formula of qRT-PCR:Be respectively compared 3 specific candidate differential expression lncRNA and Its 10 target genes are in 4 tibetan sheep groups (2 High aititude groups and 2 intermediate altitude groups) and control group (low altitude area) sheep Expression quantity in group is horizontal.As a result as shown in Figure 5: qRT-PCR stable amplification result, wherein 3 specific candidate difference tables The significant up-regulation of expression is presented in liver organization up to lncRNA, relative expression levels are between 0.6279-5.9141, hence it is evident that be higher than pair According to group (low altitude area) sheep liver organization relative expression levels；2 specific candidate differential expression lncRNA (TCONS_ 00332125 and TCONS_00377466) it is presented in lung tissue and expresses significant downward, relative expression levels are in 0.0042- Between 0.0857, hence it is evident that be lower than control group (low altitude area) sheep lung tissue relative expression levels, and 1 specific candidate difference It expresses lncRNA (TCONS_00139593) and the significant up-regulation of expression is presented in lung tissue, relative expression levels are in 2.7931- Between 4.7042, hence it is evident that be higher than control group (low altitude area) sheep lung tissue relative expression levels.

4 target genes (ENSOARG00000007330, ENSOARG00000007403, ENSOARG00000007412 and ENSOARG00000018872 the significant downward of expression) is presented in liver and lung tissue, relative expression levels exist respectively Between 0.0763-0.9654 and 0.0022-0.5571, it is significantly lower than control group (low altitude area) sheep liver and lung tissue phase To expression；2 target genes (ENSOARG00000007321 and ENSOARG00000006342) are in liver and lung tissue It is presented the significant up-regulation of expression, relative expression levels are respectively between 0.6467-1.2546 and 1.1334-4.5153, obviously Higher than control group (low altitude area) sheep liver and lung tissue relative expression levels；3 target genes (ENSOARG00000006399, ENSOARG00000007219 and ENSOARG00000000775) is in liver and lung tissue point Significant upper reconcile Cheng Xian not be expressed to lower, relative expression levels respectively between 0.6339-1.9008 and 0.0197-0.5896, Control group (low altitude area) sheep liver and lung tissue relative expression levels are apparently higher than and are lower than respectively.Therefore, the results showed that QRT-PCR result is consistent with RNA-seq result.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Sequence table

<110>Lanzhou Livestock and Animal Drug Inst., Chinese Academy of Agricultural Science

<120>application of a kind of lncRNA in the detection of tibetan sheep hypoxia adaptability

<160> 29

<170> SIPOSequenceListing 1.0

<210> 1

<211> 769

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

acgggagagt actctaactg ggtccagctt aaagcgttgt gaaaccactt cctgctagaa 60

gaagctgaac gcagtagatg aagggaagca gaacatctgg gttccatgcc agtgtcagcc 120

gtgggtaaaa ggacagaaca atgaaacggg tctttaatac attgatgaaa ggtggacgga 180

attgtgttca gatgacggga cgagccagcg ttgtgtttgg ccagatggtg ggaatctccc 240

agaaggagaa ggacacacgg ctggtgaaac tccagtgctt cagtggaccc atcagagtct 300

tgcagcctcc gtattctcat gcttccttca tggatctgca tgctgggggg tgatgtaccc 360

aatgtctcag caatcgccag tctcaaaacc acttgtgatt gttcaaacgc ccagtcacat 420

ccagctcttt gcaaccccat ggactgcagc atgccgggcc tccgtgtccc tcaccgtctc 480

ctggagtttg cccaagttca tgttcattgc atcggtgatg ccatctagcc atctcatcct 540

ctgatgccct cttctctttc tgctttcaat ctttcccagc atcagggact tttccaatga 600

atcatctgtt cacagtcaga taaccaaaat actggagctt cagcttcagc attagtcctt 660

ccagtgaata ttcagggttg atctctctta agattgactt actacttact tcaccaatta 720

acaaacttct atcccttgtg ctaattagct aatcttccaa tgtggattt 769

<210> 2

<211> 1393

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

ggattagtga aggaaggcag ctcggaatct gataccttcc tgacgtagcc accaggcaga 60

aggagacgtc tcgagtgccc gggctggggg aggcagtggc accagctctg tccccggctc 120

cccagctggg ccccaccttg ctgacagggg acaggcctgc tgcgggtcac ccacagtgcg 180

ctgggctgcc ctccaagaac ccctggctcc ccctccgccc catcggcact tccagtgact 240

ggcctcagcc tccatgccag cgcagagtca ccgccaggac ctcagcccta atttctcatc 300

tctgctccac cggccatccc ctccaagggc cagacctgga actgttctct ctggaagtcc 360

gaaggcacac cagcagcgcg cctgcagacc agcacagccc aggagacgca gcaaggaaca 420

gacagaaaag cggaagccgg cctggaagac gccctagcgg agtccagaca ctgtactgct 480

tgaaactccc cgtggagaag gcgtcccccc caccggcaaa tcaggctgcc ctggaacgct 540

ggggacggca gaatatgctg gaagaagtgc agtggtccct aggctggcct ggtgccggct 600

gggcactgtc aagtgacccc agtggatgga cagggagctg aagaaccacc caaagacttc 660

ggcccaaaat cttgactcac aacagcatga gttaaaatgg ttgctttaaa ccacttaggt 720

taggggtgat ttgctactta ggggtgactg tcgcatagca gcagataaag gaacagctgt 780

ctaacctcac tagaactcaa ggaaactcgg atggcttccc attctaaccc atcaggttgg 840

caaatgtttg acaggttaca gacgggtcta acagagagac ccttgcttac ggcggtcaga 900

ctgtcggccg acacgggggc ctggatgcga agccgacaga tctctgcaga ttttacacac 960

aggttccagg gcccagcagt tccccttaga ctaccaccct gtttactcag gcgcccccag 1020

cgatgttcct cataacggca aaccagctag gcacggcctc caggtccagc agcagggcgg 1080

atagaccagg cacgtgctca ggcagaaccc agccccagca tcaaggtgaa caagctacgg 1140

tggcgcctgg ccccgaaaaa caggccgacc cttggttgtc ccaggcgggc atgagctgct 1200

caaccttccc cacggtcggg ccacagcctc ctctgcaaac aggctgagcc ctcttcgggg 1260

accctgccag cctccgccgc accctctcac tgcagctctg gctggagagc tgcttccttc 1320

tgtcccgttt ccaaatctgt aacacctgag gaatgacgag gcccacctcc cagtgttcct 1380

gggactcaag gac 1393

<210> 3

<211> 533

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

gttcaagagc cagggaccca gagtgtgaat ctcacttctc caatcactag ccaggtcacc 60

tcagaccatg ctggccattg catggcctct gtttactcat ctgtacaatg ggcatgatca 120

atactcactg catagggcta ctggaaggac tcaagtgagc tcatggatgc acagatcatg 180

gagcccaagg tgtggagaaa gggaggctgt ggttattctc aagaggggaa tagtcacttg 240

tgtaagctct ggtgcagagg cagaaatgcc gagagatact gaggcaagtt ttcattcttt 300

gcctcattta tttagccaaa ccttttgaag ataaagaagt tagtgttttc taaaattttt 360

ggaagacctg accaattcag ttcagttcag tcactcagtc atgtccaact ctttgcaacc 420

ccatggactg cagcacgcca ggcctccctg tccatcacca actcccagag tttactcaaa 480

ctcatgtctg ttgagtcggt gatgcaatcc aaccatctca tcctctgtcg tcc 533

<210> 4

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

ctagaagaag ctgaacgcag ta 22

<210> 5

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

cgtccacctt tcatcaatgt at 22

<210> 6

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

agacactgta ctgcttgaaa c 21

<210> 7

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

ctgcacttct tccagcatat t 21

<210> 8

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

cacagatcat ggagcccaa 19

<210> 9

<211> 18

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

tgcctcagta tctctcgg 18

<210> 10

<211> 18

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

cagttcttgg caggatgg 18

<210> 11

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

tcagaggaca cttggattca c 21

<210> 12

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

ttaccagatc cctgccgaa 19

<210> 13

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

acttgactcc tctaagcct 19

<210> 14

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

ccactcggac atcaaacata tc 22

<210> 15

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

gctctaaggt cacaggtttc 20

<210> 16

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

cctgactgac cacttctatg 20

<210> 17

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 17

gatgtaggtg aaggaccgat 20

<210> 18

<211> 18

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 18

cacaaaggct tcctcacg 18

<210> 19

<211> 18

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 19

tgtttccaga gccggatg 18

<210> 20

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 20

tgatcttctc ggacgtgtat 20

<210> 21

<211> 18

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 21

tccagtgggt agtgttcg 18

<210> 22

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 22

gatgggaccc tcatgctaga 20

<210> 23

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 23

cgtcagagta gagccccttg 20

<210> 24

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 24

cggctatcga gaaggacttt a 21

<210> 25

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 25

cgggaagttc ttgggataca 20

<210> 26

<211> 18

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 26

gaggcccgga tggaagta 18

<210> 27

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 27

cacagcagct catgaagga 19

<210> 28

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 28

gcggctttac gatcatcaac ta 22

<210> 29

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 29

aggaccagtc gattccatac a 21

Claims (5)

1. Application of the 1.lncRNA in the detection of tibetan sheep hypoxia adaptability, which is characterized in that the lncRNA is TCONS_ 00332125, TCONS_00377466 or TCONS_00139593；Described TCONS_00332125, TCONS_00377466 and The sequence of TCONS_00139593 is respectively as shown in SEQ IDNO.1-3.
2. 2. the kit of hypoxia adaptability in a kind of detection tibetan sheep liver and lung tissue, which is characterized in that including with the following group Point: the reverse transcription system of total serum IgE and the amplification system of cDNA；It include total serum IgE, reverse transcription in the reverse transcription system of the total serum IgE Enzyme, dNTP and oligdT；It include TCONS_00332125 primer, TCONS_00377466 primer in the amplification system of the cDNA With TCONS_00139593 primer, the nucleotide sequence of the TCONS_00332125 primer such as SEQ ID NO.4 and SEQ ID Shown in NO.5, nucleotide sequence nucleosides as shown in SEQ ID NO.6 and SEQ ID NO.7 of the TCONS_00377466 primer Acid sequence, the nucleotide sequence of the TCONS_00139593 primer is as shown in SEQ ID NO.8 and SEQ ID NO.9；It is described The sequence of TCONS_00332125, TCONS_00377466 and TCONS_00139593 are as shown in SEQ IDNO.1-3.
3. 3. the application method of kit described in claim 2, which comprises the following steps:

   Extract test serum sample total serum IgE；

   The total serum IgE reverse transcription of extraction is prepared into cDNA using the total serum IgE reverse transcription system；

   Using the cDNA amplification system, with the TCONS_00332125 primer, TCONS_00377466 primer and TCONS_ The cDNA that 00139593 primer pair is prepared carries out qRT-PCR and expands lncRNA.
4. Application of the 4.lncRNA in target gene detection, which is characterized in that the lncRNA is TCONS_00332125, TCONS_ 00377466 or TCONS_00139593, described TCONS_00332125, TCONS_00377466 and TCONS_00139593's Sequence is respectively as shown in SEQ ID NO.1-3；The prediction of the hypoxia adaptability target gene passes through cis the and trans mode of action It realizes；The target gene be ENSOARG00000007330, ENSOARG00000007403, ENSOARG00000007412, ENSOARG00000018872、ENSOARG00000007321、ENSOARG00000006342、ENSOARG00000006399、 ENSOARG00000007219 and ENSOARG00000000775.
5. 5. application according to claim 4, which is characterized in that the target gene of the hypoxia adaptability lncRNA ENSOARG00000006342 qRT-PCR amplimer is SEQ ID NO.10 and SEQ ID NO.11；

   The target gene ENSOARG00000006399 qRT-PCR amplimer of the hypoxia adaptability lncRNA is SEQ ID NO.12 and SEQ ID NO.13；

   The target gene ENSOARG00000007219 qRT-PCR amplimer of the hypoxia adaptability lncRNA is SEQ ID NO.14 and SEQ ID NO.15；

   The target gene ENSOARG00000007321 qRT-PCR amplimer of the hypoxia adaptability lncRNA is SEQ ID NO.16 and SEQ ID NO.17；

   The target gene ENSOARG00000007330 qRT-PCR amplimer of the hypoxia adaptability lncRNA is SEQ ID NO.18 and SEQ ID NO.19；

   The target gene ENSOARG00000007403 qRT-PCR amplimer of the hypoxia adaptability lncRNA is SEQ ID NO.20 and SEQ ID NO.21；

   The target gene ENSOARG00000007412 qRT-PCR amplimer of the hypoxia adaptability lncRNA is SEQ ID NO.22 and SEQ ID NO.23；

   The target gene ENSOARG00000018872 qRT-PCR amplimer of the hypoxia adaptability lncRNA is SEQ ID NO.24 and SEQ ID NO.25；

   The target gene ENSOARG00000000775 qRT-PCR amplimer of the hypoxia adaptability lncRNA is SEQ ID NO.26 and SEQ ID NO.27.