CN108330170A - A kind of application of lncRNA in the detection of tibetan sheep hypoxia adaptability - Google Patents

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

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CN108330170A
CN108330170A CN201710885564.1A CN201710885564A CN108330170A CN 108330170 A CN108330170 A CN 108330170A CN 201710885564 A CN201710885564 A CN 201710885564A CN 108330170 A CN108330170 A CN 108330170A
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lncrna
tcons
sheep
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刘建斌
丁学智
曾玉峰
郭宪
袁超
郭婷婷
孙晓萍
岳耀敬
梁春年
褚敏
冯瑞林
牛春娥
郭健
杨博辉
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Lanzhou Institute of Animal Husbandry and Veterinary Medicine CAAS
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Abstract

The present invention provides applications 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 spectras, screening is through qRT PCR verifications and the relevant lncRNA of hypoxia adaptability, 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 provide science support 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 technology
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 to the north of Nyainqentanglha Shan and Yarlung Zangbo River area；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).It hides 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 pattern, 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, find tibetan sheep unit area alveolar number it is more notable than Small-fat-tail sheep increase, the thickness of alveolar septa dramatically increases, The caliber of bronchioli terminales is substantially reduced, and thinks that these differences and feature are the shapes that tibetan sheep lung tissue adapts to Alpine cold and hypoxia 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 inhibitors, tibetan sheep pulmonary arterial pressure has 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 objects, 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 genes in High aititude sheep placenta raises, and body alleviates hypoxemia by synthesizing 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, packed cell volume, 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 are metabolized 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 enzymes 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 Go out complicated response (Wu, 2012).
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 genes regulate and control net in High aititude hypoxemia Central role is played in network, at the same also contain with the relevant three Gene A dam17, Arg2 of 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 fully 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 The Transform efficiency of diet dry matter, crude protein, digestibility of fiber, nitrogen retention, daily ration nitrogen use efficiency, effective urea (Huang andLiu,2009；Zhou Jianwei, 2015), disclose the lower nutrition flexibility mechanism of tibetan sheep nitrogen stress.
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 stablized 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 finds that 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 silence, 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, also tibetan sheep hypoxia adaptability correlation work(is not carried out using lncRNA The method of energy genetic test.
Invention content
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 provides following technical scheme：
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 includes the following steps：
1) the Different Altitude tibetan sheep that there are 3 biology to repeat and control group sheep liver and lung tissue sample are extracted LncRNA sequencings are 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 sheeps.
Preferably, the step 1) includes the following steps：
1.1) total serum IgE of tibetan sheep and sheep liver and lung tissue sample is extracted；
1.2) cDNA library of step 1.1) the extraction total serum IgE is built；
1.3) cDNA library that step 1.2) described in quality inspection is built；
1.4) machine on the cDNA library of the total serum IgE is sequenced, Clean Data data is obtained by analysis.
Preferably, the step 2) includes the following steps：
2.1) the Clean Data data are pre-processed and is uniformed, so that Q30 base percentages is more than 85%, obtain Obtain valid data；
2.2) valid data for obtaining the step 2.1) carry out sequence alignment with reference gene group Oar_v3.1, obtain Mapped Reads；
2.3) the Mapped Reads are subjected to transcript splicing using Cufflinks and Scripture programs；
2.4) transcript for selecting length >=200bp, Exon number >=2, each transcript is calculated 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；The reverse transcription system of the total serum IgE includes total serum IgE, inverse Transcriptase, dNTP and oligdT；The amplification system of the cDNA includes TCONS_00332125 primers, TCONS_00377466 Primer and TCONS_00139593 primers, the TCONS_00332125 primers have SEQ ID NO.4 and SEQ ID NO.5 institutes Show that nucleotide sequence, the TCONS_00377466 primers have nucleotides sequence shown in SEQ ID NO.6 and SEQ ID NO.7 Row, the TCONS_00139593 primers have nucleotide sequence shown in SEQ ID NO.8 and SEQ IDNO.9.
The present invention also provides the application methods of the kit, include 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 primers, TCONS_00377466 primers and TCONS_00139593 primer pairs 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 modes of action Predict 10 target genes of the lncRNA.
Preferably, the qRT-PCR amplimers of the hypoxia adaptability lncRNA target genes include SEQ ID NO.10- 29。
Beneficial effects of the present invention
LncRNA of the present invention includes TCONS_00332125, TCONS_00377466 or TCONS_00139593, The detection of tibetan sheep hypoxia adaptability is can be applied to, helps 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 of a group research offer science support.
Description of the drawings
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 results of liver and lung tissue tibetan sheep hypoxia adaptability lncRNA and its target gene.
Specific implementation mode
It is described the present invention provides applications 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 sequencings, 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 It is detected hypoxia adaptability lncRNA to select lncRNA.
The Different Altitude tibetan sheep specifically includes High aititude bar tibetan sheep and A Wang tibetan sheeps suddenly in the present invention；Institute State that a bar tibetan sheep height above sea level suddenly is 4468m, the A Wang tibetan sheeps 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 primers have SEQ ID NO.4 and SEQ ID Nucleotide sequence shown in NO.5, the TCONS_00377466 primers have nucleosides shown in SEQ ID NO.6 and SEQ ID NO.7 Acid sequence, the TCONS_00139593 primers 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 companiesReagent carries out, and specific operating procedure is shown inReagent article No. is 15596026 specification.
The present invention preferably uses 2000 spectrophotometers of NanoDrop after extraction obtains 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 meets above-mentioned in the total serum IgE purity and concentration for determining extraction 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 The cDNA library of RNA is built, the construction cDNA library specifically includes the following steps：
1.21) rRNA in epicentre Ribo-ZeroTM kits removal sample total serum IgE is utilized, rRNA- is obtained DepletedRNA, specific steps are operated according to epicentre Ribo-ZeroTM kit specifications；
1.22) Fragmentation Buffer and then into the rRNA-depleted RNA are added, by rRNA- Depleted RNA are 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 solution, dATP, DUTP, dCTP, dGTP, RNase H and DNApolymerase I synthesize Article 2 cDNA chains, utilize AMPure XPbeads couple The double stranded cDNA purification of synthesis；
1.24) end reparation plus A are carried out successively to the double-strand cDNA of the purifying and connects sequence measuring joints, use AMPure XPbeads selects the segment of 180-200bp sizes；
1.25) the double-strand cDNA degradations chain containing U for obtaining the step 1.24), is enriched with to obtain cDNA library by PCR.
The present invention preferably carries out library Quality Control, library matter after the cDNA library for obtaining RNA to the cDNA library of acquisition Machine sequencing is carried out after control is qualified, Clean Data are obtained by analysis.The library Quality Control preferably uses Qubit 2.0 The concentration and Insert Fragment size (Insert Size) in library are detected with Agilent 2100, Insert Fragment size is excellent Choosing is 180-200bp；Accurate quantitative analysis is carried out to the effective concentration in library using Q-PCR methods, the effective concentration in library is more than 2nM is qualification.
The present invention carries out machine sequencing after library Quality Control qualification, and the sequencing preferably carries out high pass with HiSeq2500 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.Clean Data data are analyzed in the present invention to include the following steps：
2.1) Clean Data data are pre-processed and is uniformed, so that Q30 base percentages is more than 85%, had Imitate data；
2.2) valid data for obtaining step 2.1) carry out sequence alignment with reference gene group Oar_v3.1, obtain Mapped Reads；
2.3) Mapped Reads are subjected to transcript splicing using Cufflinks and Scripture programs；
2.4) transcript for selecting length >=200bp, Exon number >=2, each transcript is calculated 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 primers of the lncRNA are designed with primer-design software according to lncRNA sequences Synthesis, TCONS_00332125 upstream and downstream primers are respectively SEQ ID NO.4 and SEQ in the preferred lncRNA ID NO.5, TCONS_00377466 upstream and downstream primers 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 verification detects hypoxia adaptability lncRNA and lung tissue sample are total RNA steps extract 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 The preparation of product cDNA is preferably：Reverse transcription synthetic tissue sample cDNA is carried out 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
Table 1 removes the reaction system of genomic DNA
Reagent Usage amount
4×gDNAwiperMix 2.0μL
TotalRNA 0.5μg
Nuclease-freeH2O Add to 8.0 μ L
The first step is reacted required mentioned reagent mixing and is placed on 42 DEG C of reaction 2min by the present invention.
Reaction system such as table 2 by second step reverse transcription synthesis cDNA in the present invention
2 reverse transcription of table synthesizes the reaction system of 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 pairs cDNA of lncRNA Increase.It is specific in the present invention to useGreen PCRKit kits (Qiagen, Germany) QRT-PCR amplifications are carried out, are existed as template using the cDNA of above-mentioned acquisition480 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℃5min；95 DEG C of 10s, 60 DEG C of 30s, 40 cycles；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 applications 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 Cis the and trans modes of action are crossed to interact with its target gene.The Cis modes of action are 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, preferably opens 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 to promote expression.
The expression quantity of heretofore described hypoxia adaptability lncRNA and its target gene is tested by qRT-PCR amplifications Card.The method and steps of the expression quantity qRT-PCR amplifications of heretofore described hypoxia adaptability lncRNA is with above-mentioned lncRNA's QRT-PCR detection methods are consistent, and details are not described herein.The qRT-PCR amplifications of the hypoxia adaptability lncRNA target genes are drawn Object includes preferably 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 the Adaptive mechanism under tibetan sheep coerces low-oxygen environment, enriches extreme environment Basic theory in terms of ruminant molecular ecology adaptability, and provided for tibetan sheep molecular breeding and functional genomics research Science supports.
Present invention will be further explained below with reference to specific examples, is only used for explaining the present 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 the 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 companiesReagent (article No. 15596026) specification extract liver and The Total RNA of lungs, then extracted with 2000 spectrophotometers of NanoDrop (Thermo Scientific, USA) measurement RNA purity and concentration, agarose gel electrophoresis quality inspection ensure extraction RNA integrality.
2.2RNA library construction
2.2.1 epicentre Ribo-ZeroTM kits removal sample rRNA is utilized；
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 solution, dATP, dUTP, dCTP, dGTP, RNase H and DNApolymerase I synthesis is added in first cDNA chain Article 2 cDNA chains purify cDNA using AMPure XP beads；
2.2.4 the double-strand cDNA purified carries out end reparation plus A and connects sequence measuring joints again, 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 analysis
3.1 sequencing datas and its quality control
It is controlled by sequencing quality, obtains 379Gb CleanData altogether, to the Clean Data data that are obtained into line number Data preprocess and homogenization, each sample Q30 base percentages reach 85% or more.
3.2 long-chain non-coding data and reference gene group sequence alignment
It is analyzed using genome Oar_v3.1 as with reference to progress sequence alignment and follow-up lncRNA, Oar_v3.1 downloads ground Location：//ftp.ensembl.org/pub/release-78/fasta/ovis_aries/。
3.3 long-chain non-coding Library Qualities are assessed
Qualified long-chain non-coding library is the necessary condition of long-chain non-coding sequencing, to ensure the quality in library, from Lower 3 different angles carry out quality evaluation to long-chain non-coding sequencing library：
3.3.1 by examining distributions of the Mapped Reads on transcript, randomness, the mRNA of mRNA fragmentations 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.
3.4 transcripts splice
Mapped Reads are subjected to transcript splicing using Cufflinks and Scripture programs.
3.5 lncRNA are screened
LncRNA basic screenings include：Remove the mRNA (transcript and its spliceosome) in genome database, selection length The transcript of degree >=200bp, Exon number >=2 calculates the minimum vertex-covering degree of each transcript using cufflinks, selects 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 detections.
4, result
4.1 the present invention using Cufflinks and Scripture assembling obtain 473766 transcripts, by CPC, PFAM, CNCI, CPAT encode potency analysis, are identified in liver organization and obtain 8107 lncRNA (Fig. 1), 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 shares 2 Hypoxia adaptation specificity with control group sheep liver organization 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 modes 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：For dye class qRT- The primer 13 of PCR verifications 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 kits 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 kits (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℃5min；95 DEG C of 10s, 60 DEG C of 30s, 40 cycles；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.The results are shown in Figure 5：QRT-PCR stable amplification results, wherein 3 specific candidate difference tables Up to lncRNA liver organization present expression significantly up-regulation, relative expression levels are between 0.6279-5.9141, hence it is evident that 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 presents to express in lung tissue and significantly lower, relative expression levels are in 0.0042- Between 0.0857, hence it is evident that be less than control group (low altitude area) sheep lung tissue relative expression levels, and 1 specific candidate difference It expresses lncRNA (TCONS_00139593) and expression significantly up-regulation 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 expression) is presented in liver and lung tissue significantly to lower, 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 Expression significantly up-regulation is presented, 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 Significantly 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 less than respectively.Therefore, the results showed that QRT-PCR results are consistent with RNA-seq results.
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>A kind of application 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 (9)

  1. Applications 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 as shown in SEQ IDNO.1-3.
  2. 2. applying according to claim 1, which is characterized in that the screening technique of the lncRNA includes the following steps：
    1) the Different Altitude tibetan sheep that there are 3 biology to repeat and control group sheep liver and lung tissue sample are extracted respectively LncRNA sequencings are carried out, Clean Data data are obtained by analysis；
    2) lncRNA that the Clean Data data obtain prediction is analyzed；
    3) it is respectively compared the time that significant difference is expressed in Different Altitude tibetan sheep and control group sheep liver and lung tissue sample It is detected hypoxia adaptability lncRNA to select lncRNA.
  3. 3. applying according to claim 2, which is characterized in that the Different Altitude tibetan sheep includes High aititude bar tibetan sheep suddenly With the white tibetan sheep of A Wang tibetan sheeps and intermediate altitude Qilian and Gan Jia tibetan sheeps.
  4. 4. applying according to claim 2, which is characterized in that the step 1) includes the following steps：
    1.1) total serum IgE of tibetan sheep and sheep liver and lung tissue sample is extracted；
    1.2) build the cDNA library that the step 1.1) extracts total serum IgE；
    1.3) cDNA library that step 1.2) described in quality inspection is built；
    1.4) machine on the cDNA library of the total serum IgE is sequenced, Clean Data data is obtained by analysis.
  5. 5. applying according to claim 2, which is characterized in that the step 2) includes the following steps：
    2.1) the Clean Data data are pre-processed and is uniformed, so that Q30 base percentages is more than 85%, had Imitate data；
    2.2) valid data for obtaining the step 2.1) carry out sequence alignment with reference gene group Oar_v3.1, obtain Mapped Reads；
    2.3) the Mapped Reads are subjected to transcript splicing using Cufflinks and Scripture programs；2.4) it selects The transcript of length >=200bp, Exon number >=2 calculates minimum vertex-covering degree, the selection of each transcript using cufflinks The transcript of minimum vertex-covering degree >=3, or the lncRNA spliced simultaneously by Cufflinks and Scripture is prediction lncRNA。
  6. 6. 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；The reverse transcription system of the total serum IgE includes total serum IgE, reverse transcription Enzyme, dNTP and oligdT；The amplification system of the cDNA includes TCONS_00332125 primers, TCONS_00377466 primers With TCONS_00139593 primers, the TCONS_00332125 primers have core shown in SEQ ID NO.4 and SEQ ID NO.5 Nucleotide sequence, the TCONS_00377466 primers have nucleotide sequence shown in SEQ ID NO.6 and SEQ ID NO.7, institute TCONS_00139593 primers are stated with nucleotide sequence shown in SEQ ID NO.8 and SEQ ID NO.9.
  7. 7. the application method of kit described in claim 6, which is characterized in that include 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 systems, with the TCONS_00332125 primers, TCONS_00377466 primers and TCONS_ The cDNA that 00139593 primer pair is prepared carries out qRT-PCR and expands lncRNA.
  8. Applications of the 8.lncRNA in target gene detection, which is characterized in that the lncRNA is TCONS_00332125, TCONS_ The prediction of 00377466 or TCONS_00139593, the target gene are realized by cis the and trans modes of action.
  9. 9. application according to claim 8, which is characterized in that the target gene qRT-PCR of the hypoxia adaptability lncRNA Amplimer includes SEQ ID NO.10-29.
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