CN107586855A - A kind of applications of mRNA in the detection of tibetan sheep hypoxia adaptability - Google Patents
A kind of applications of mRNA in the detection of tibetan sheep hypoxia adaptability Download PDFInfo
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Abstract
The invention provides a kind of mRNA tibetan sheep hypoxia adaptability detection in application, the mRNA detection method and detection kit；The present invention passes through system research tibetan sheep hypoxia adaptability mRNA express spectras, the mRNA related to hypoxia adaptability that screening is verified through qRT PCR, tibetan sheep hypoxia adaptability can be applied to detect, 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
Technical field
The invention belongs to technical field of molecular biology, and in particular to a kind of mRNA is in the detection of tibetan sheep hypoxia adaptability
Application.
Background technology
Qinghai-Tibet tibetan sheep is broadly divided into plateau type (Grassland Type), mountain valley-type and Euler's type three major types, 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 is distributed in, due to each ecological condition most diverse, is 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；The tibetan sheep in Qinghai is mainly distributed on Haibeitibetan autonomy
State, Tibetan Autonomous Prefecture of Hainan, Kazak autonomous prefecture of the Hai Xi Mongols, Tibetan Autonomous Prefecture of Huangnan, Tibetan Autonomous Prefecture of Yushu, Golog are hidden
The wide Alpine-arctic Pastoral in the state of autonomous prefecture of race six；The tibetan sheep in Gansu is mainly distributed on each counties and cities of Tibetan Autonomous Prefecture of Gannan；Sichuan
Domestic tibetan sheep is mainly distributed on Tibetan Autonomous Prefecture of Garze, Abazangzuqiangzu Autonomous Prefecture the north pastoral area.Tibetan sheep
Banma, some areas in the county of Nangqian two of Southern Qinghai Province are mainly distributed on, Sichuan Province is herded in Abazangzuqiangzu Autonomous Prefecture south
Area, Zhaotong City, Qujing, Lijiang City and the Baoshan City Tengchong County in Yunnan.Euler's type tibetan sheep is a special ecological type, in
Heart producing region is located at Maqu County Euler township and neighbour area and the autonomy of Qinghai Province's Henanmongol of Gansu Province Tibetan Autonomous Prefecture of Gannan
The ground such as county 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 uniqueness hypoxia adaptability strategy (Liu et al., 2016；Wei et al.,2015；Beall,2013).Hide
Sheep is the indigenous ruminant domestic animal of Qinghai-Tibet Platean and the important component of Grassland ecosystems, is the important life of Tibetan people
And the means of production, while be also the symbol (Long et al., 2008) of its wealth.Tibetan sheep herds pipe in extensive and traditional
Under reason pattern, ecological environment harsh to Qinghai-Tibet Platean over the past thousands of years has extremely strong adaptability.Tibetan sheep passes through long-term
Natural selection and evolution, have to 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 (Guo et al., 2012；Wang et al.,2011).
In studying the lung tissue structure of High aititude tibetan sheep and low altitude area Small-fat-tail sheep, find tibetan sheep unit area alveolar number than small
Tail is cold, and sheep is significantly increased, the thickness of alveolar septa is dramatically increased, the caliber of bronchioli terminales is substantially reduced, and thinks these differences
It is the Morphological Characteristics (Yu Hongxian, 1999) that tibetan sheep lung tissue adapts to Altitude with feature.Determine the pulmonary artery of tibetan sheep
Pressure, PAWP, body arterial pressure and cardiac output, pulmonary vascular resistance is calculated, in the presence of NOS non-selective inhibitors,
Tibetan sheep pulmonary arterial pressure has increased slightly, cardiac output reduce, and 4500 meters than 2300 meters at tibetan sheep pulmonary vascular resistance it is notable
Increase (P<0.05)；Under the effect of NOS selective depressions thing, only the former pulmonary vascular resistance increase, the latter do not change.With
Endogenous NO S activity is suppressed, and NO growing amount is also reduced, and promotes body Pulmonary Vascular to shrink, and tibetan sheep is adapted to hypoxemia ring
Border (Ruan et al., 2004).The measurement result of this pulmonary vascular resistance with Koizumi etc. to Different Altitude sheep is consistent
(Koizumi et al.,2004).Under the conditions of chronic hypoxia, VEGF the and NOS genes in High aititude sheep placenta are introduced
Expression quantity raises, and body alleviates hypoxemia by synthesizing VEGF, and this is probably long-term habit of the low altitude area sheep in high altitude localities
Take mechanism (Parraguez et al., 2010).In to High aititude tibetan sheep and the research of low altitude area sheep biochemical indicator, hide continuous
Sheep biochemical indicator (red blood cell, packed cell volume, mean corpuscular volume (MCV), mean corpuscular hemoglobin) is high or significantly high,
Adaptation of the tibetan sheep to hypoxemia is not to be realized from increase hemoglobin concentration, it may be possible to improves hemoglobin conveying
The ability and efficiency of oxygen, these unique physiological functions are advantageous to increase pulmonary ventilation volume and pulmonary blood flow volume, and pass through endogenous NO
Yield height adjusts pulmonary blood vasculature to adapt to the environment of hypoxemia (Liu et al., 2016).
Tibetan sheep is not simply formed with effect intake, conveying and the architectural 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 environment of low oxygen plateau (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 change of some adaptability related genes such as transmission, response anoxic, DNA repair enzymes and signal path；In low-oxygen environment
Histocyte then hypoxia signal may be experienced by the oxygen acceptor molecule on its film, and start many hypoxic effect genes and do
Go out the response (Wu, 2012) of complexity.In recent years the god of low-oxygen environment is adapted to the innovation of technique of gene detection, high and cold indigenous animal
Secret veil is progressively opened.By carrying out genome-wide screening to the native sheep breeds 122 of Different Altitude gradient 7 individual,
236 differential genes and 8 specific candidate genes for adapting to low-oxygen environment are disclosed, wherein EPAS1 genes are in High aititude hypoxemia
Central role is played in regulated and control network, at the same also contains the three Gene A dam17s related to hypoxia inducible factor HIF-1 α,
Arg2、Mmp3(Wei et al.,2016；Liu et al.,2016).Utilize high throughput sequencing technologies and analysis of biological information plan
Slightly, fully disclosed from genomic level Tibetan pig (Li et al., 2013) and titmouse (Qu et al., 2013) it is peculiar
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, the Transform efficiency of effective urea
(Huang and Liu,2009；Zhou Jianwei, 2015), disclose the lower nutrition flexibility mechanism of tibetan sheep nitrogen stress.
However, tibetan sheep is distinctive, ancient, the best in quality carpet wool sheep local group in China Qinghai-Tibet Platean.
Because tibetan sheep can adapt to severe puna environment, make full use of other unserviceable natural resources of big domestic animal.Cause
It lives in that hypoxemia, cold, drying, radiation be strong, little precipitation for a long time, the highlands that 4000 meters of mean sea level, in environment pressure
Under power and extensive style feeding manner, formed to hypoxemia, dried the stable genetic mechanism in habitat.
The special mark that searching natural selection and artificial selection pressure leave on tibetan sheep genome, identify selection signal
The important regulatory pathway occurred where adaptive mutation gene and these genes in region, so as to annotate on a molecular scale
Unique hypoxia adaptability feature of the tibetan sheep formed in very long evolutionary process, tibetan sheep is disclosed under low-oxygen environment stress
Adaptive mechanism.This is the purpose that those skilled in the art study for many years, still, under prior art is coerced low-oxygen environment
Adaptive mechanism is not known, without any report on mRNA in terms of tibetan sheep hypoxia adaptability.
The content of the invention
In view of this, it is an object of the invention to provide a kind of tibetan sheep liver organization and lung tissue hypoxia adaptability
Application in mRNA detections.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The invention provides a kind of applications of mRNA in the detection of tibetan sheep hypoxia adaptability, the mRNA is
ENSOARG00000002655、ENSOARG00000005252、ENSOARG00000007592、ENSOARG00000008263、
ENSOARG00000008620、ENSOARG00000009196、ENSOARG00000010493、ENSOARG00000010939、
ENSOARG00000014554, ENSOARG00000014659 and ENSOARG00000014870.
Present invention also offers the screening technique of the mRNA, comprise the following steps：
1) the Different Altitude tibetan sheep and control group sheep liver and lung tissue sample that extraction repeats with 3 biology
MRNA sequencings are carried out, Clean Data data are obtained by analyzing；
2) the Clean Data data are analyzed and obtains Different Altitude tibetan sheep and control group sheep differential expression mRNA；
3) time that significant difference is expressed in Different Altitude tibetan sheep and control group sheep liver and lung tissue sample is screened
It is the hypoxia adaptability mRNA for detecting and obtaining to select mRNA.
Preferably, the Different Altitude tibetan sheep includes High aititude bar tibetan sheep and A Wang tibetan sheeps and intermediate altitude Qi suddenly
Even white tibetan sheep and Gan Jia tibetan sheeps.
Preferably, the step 1) comprises the following steps：
1.1) tibetan sheep and the total serum IgE of control group sheep liver and lung tissue sample are 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 qualified cDNA library of quality inspection is sequenced, obtains 379Gb Clean Data data altogether.
Preferably, the step 2) comprises the following steps：
2.1) the Clean Data data are pre-processed and uniformed, Q30 bases percentage is more than 85%, obtain
Obtain valid data；
2.2) valid data that the step 2.1) obtains and reference gene group Oar_v3.1 are subjected to sequence alignment, obtained
Mapped Reads；
2.3) the Mapped Reads are spliced using Cufflinks programs；Carried out with genome annotation information
Comparative analysis, assembling obtain transcript；
2.4) excavated in transcript and obtain differential expression mRNA.
Preferably, after obtaining the differential expression mRNA, functional analysis is carried out to differential expression mRNA.
Preferably, the functional analysis includes functional annotation, enrichment and interactions between protein network analysis.
Preferably, the enrichment analysis includes GO databases and KEGG databases using database.
Present invention also offers a kind of kit for detecting hypoxia adaptability mRNA in tibetan sheep liver and lung tissue, bag
Include following components：The reverse transcription system of total serum IgE and cDNA amplification system；The reverse transcription system of the total serum IgE is included always
RNA, reverse transcriptase, dNTP and oligdT；The amplification system of the cDNA includes mRNA primers：
ENSOARG00000002655、ENSOARG00000005252、ENSOARG00000007592、ENSOARG00000008263、
ENSOARG00000008620、ENSOARG00000009196、ENSOARG00000010493、ENSOARG00000010939、
ENSOARG00000014554, ENSOARG00000014659 and ENSOARG00000014870 primer, above-mentioned mRNA's draws
Thing sequence is followed successively by nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 institutes
Show nucleotide sequence, nucleotide sequence shown in SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8
Shown nucleotide sequence, nucleotide sequence shown in SEQ ID NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID
Nucleotide sequence shown in NO.12, nucleotide sequence shown in SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15 and
Nucleotide sequence shown in SEQ ID NO.16, nucleotide sequence shown in SEQ ID NO.17 and SEQ ID NO.18, SEQ ID
Nucleotide sequence shown in NO.19 and SEQ ID NO.20, nucleotide sequence shown in SEQ ID NO.21 and SEQ ID NO.22.
Present invention also offers the application method of the kit, comprise the following steps：
Extract test serum sample total serum IgE；Using the total serum IgE reverse transcription system prepared by the total serum IgE reverse transcription of extraction
Into cDNA；Using the cDNA amplification systems, the cDNA being prepared with the mRNA primer pairs carries out qRT-PCR amplifications.
Beneficial effects of the present invention：Screening technique of the present invention can accurately filter out tibetan sheep hypoxia adaptability
MRNA, the mRNA can be applied to the detection of tibetan sheep hypoxia adaptability, while can illustrate Qinghai-Tibet tibetan sheep to hypoxemia ring
Adaptive mechanism under the stress of border, enriches the basic theory in terms of extreme environment ruminant molecular ecology adaptability, and can
Science support is provided for tibetan sheep molecular breeding and functional genomics research.
Brief description of the drawings
Fig. 1 is that the specific difference screened in Different Altitude tibetan sheep liver organization expresses mRNA；
Fig. 2 is that the specific difference screened in Different Altitude tibetan sheep lung tissue expresses mRNA；
Fig. 3 is that the specific difference screened in A Wang tibetan sheeps liver and lung tissue expresses mRNA；
Fig. 4 expresses mRNA for the specific difference screened in bar tibetan sheep liver suddenly and lung tissue；
Fig. 5 is that the specific difference screened in the white tibetan sheep liver in Qilian and lung tissue expresses mRNA；
Fig. 6 is that the specific difference screened in sweet plus tibetan sheep liver and lung tissue expresses mRNA；
Fig. 7 is the detection that liver and lung tissue specific difference express mRNA.
Embodiment
The invention provides applications of the mRNA in the detection of tibetan sheep hypoxia adaptability, the mRNA is
ENSOARG00000002655、ENSOARG00000005252、ENSOARG00000007592、ENSOARG00000008263、
ENSOARG00000008620、ENSOARG00000009196、ENSOARG00000010493、ENSOARG00000010939、
ENSOARG00000014554, ENSOARG00000014659 and ENSOARG00000014870.
The screening technique of the mRNA, comprises the following steps：
1) Different Altitude tibetan sheep and control group sheep liver are extracted respectively and lung tissue sample carries out mRNA sequencings, lead to
Cross analysis and obtain Clean Data data；
2) the Clean Data data are analyzed and obtains Different Altitude tibetan sheep and control group sheep differential expression mRNA；
3) time that significant difference is expressed in Different Altitude tibetan sheep and control group sheep liver and lung tissue sample is screened
It is the hypoxia adaptability mRNA for detecting and obtaining to select mRNA.
Different Altitude tibetan sheep described in the present invention specifically includes High aititude bar tibetan sheep and A Wang tibetan sheeps suddenly；Institute
State a bar tibetan sheep height above sea level suddenly be 4468m, the A Wang tibetan sheeps height above sea level be 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.
In the present invention, step 1) is extraction Different Altitude tibetan sheep and control group sheep liver and lung tissue sample respectively
Product carry out mRNA sequencings, and Clean Data data are obtained by analyzing.
Step 1.1) of the present invention is extraction tibetan sheep and the total serum IgE of control group sheep liver and lung tissue sample.At this
In invention, the liver and lung tissue of above-mentioned each tibetan sheep sample are preferably gathered from different place, extract respectively liver and
Lung tissue total serum IgE.The extraction of heretofore described liver and lung tissue total serum IgE preferably uses Life
Technologies companiesReagent is carried out, and specific operating procedure is shown inReagent article No. is
15596026 specification.
The present invention preferably uses NanoDrop2000 spectrophotometrics after extraction obtains liver and lung tissue total serum IgE
Count (Thermo Scientific, USA) and determine extracted total serum IgE purity and concentration, determine the OD of total serum IgE260/OD280
Between 1.8-2.1, the concentration of total serum IgE is more than or equal to 100ng/ μ L.The present invention is it is determined that the total serum IgE purity and concentration of extraction meet
After above-mentioned condition, then the total serum IgE integrality of extraction is detected, the detection of the total serum IgE integrality preferably uses agar
There are clear two bands (28S/18S rRNA), have third strip (5S sometimes in the method for sugared gel electrophoresis quality inspection, electrophoresis
RRNA), the top not may occur in which DNA pollution band, and 28S/18S should be approximate twice.
The present invention enters after purity, concentration and the integrality of preferred determination liver and lung tissue total serum IgE all meet the requirements
Row step 1.2) builds the cDNA library of step 1.1) the extraction total serum IgE；The construction cDNA library specifically includes following
Step：
1.21) rRNA in sample total serum IgE is removed using epicentre Ribo-ZeroTM kits, obtains rRNA-
Depleted RNA, specific steps are according to epicentre Ribo-ZeroTMKit specification is operated；
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 add buffer solution, dATP,
DUTP, dCTP, dGTP, RNase H and DNA polymerase I synthesis Article 2 cDNA chains, utilize AMPure XP beads
To the double stranded cDNA purification of synthesis；Using the conventional PCR in this area, the amount of raw material needed for the synthesis uses for the synthesis
The conventional dosage in this area.
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
XP beads select the fragment of 180-200bp sizes；
1.25) the segment degradation chain containing U for obtaining the step 1.24), it is enriched with to obtain cDNA library by PCR.
The present invention preferably carries out library Quality Control, library matter after RNA cDNA library is obtained to the cDNA library of acquisition
Control it is qualified after carry out machine sequencing, pass through to analyze and obtain Clean Data.Described library Quality Control preferably uses Qubit2.0
The concentration and Insert Fragment size (Insert Size) in library are detected with Agilent 2100, Insert Fragment size is excellent
Choosing for 180-200bp；Accurate quantitative analysis is carried out to the valid density in library using Q-PCR methods, the valid density in library is more than
2nM is qualified.
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 Clean Data.
The present invention is analyzed Clean Data data after Clean Data data are obtained, and obtains mRNA.The present invention
Middle analysis Clean Data data comprise the following steps：
2.1) Clean Data data are pre-processed and uniformed, Q30 bases percentage is more than 85%, had
Imitate data；
2.2) valid data that step 2.1) obtains and reference gene group Oar_v3.1 are subjected to sequence alignment, obtained
Mapped Reads；
2.3) Mapped Reads are subjected to transcript splicing using Cufflinks and Scripture programs；
2.4) excavated in transcript and obtain mRNA.
The present invention detects differential expression mRNA, in differential expression mRNA detection process, by Fold after mRNA is obtained
Change >=2 and FDR<0.02 is used as screening criteria.Differential expression analysis between sample sets is carried out using DESeq softwares, obtained
Difference expression gene collection between any two groups of difference samples.
After the present invention filters out differential expression mRNA, by Different Altitude tibetan sheep respectively with control group sheep liver and lungs
MRNA is compared in tissue, the significant difference table in Different Altitude tibetan sheep and control group sheep liver and lung tissue sample
The candidate mRNA reached is the hypoxia adaptability mRNA for detecting and obtaining.
Present invention also offers a kind of kit for detecting tibetan sheep hypoxia adaptability, including following components：Total serum IgE it is anti-
The amplification system of transcription system and cDNA；The reverse transcription system of the total serum IgE includes total serum IgE, reverse transcriptase, and qRT-PCR draws
Thing, dNTP and oligdT.
In the present invention, the qRT-PCR primers of the mRNA are to be designed to synthesize according to mRNA sequence primer-design software,
The preferable mRNA：ENSOARG00000002655、ENSOARG00000005252、ENSOARG00000007592、
ENSOARG00000008263、ENSOARG00000008620、ENSOARG00000009196、ENSOARG00000010493、
ENSOARG00000010939, ENSOARG00000014554, ENSOARG00000014659 and ENSOARG00000014870
Primer, above-mentioned mRNA primer sequence is followed successively by nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO.2, SEQ ID
Nucleotide sequence shown in NO.3 and SEQ ID NO.4, nucleotide sequence shown in SEQ ID NO.5 and SEQ ID NO.6, SEQ ID
Nucleotide sequence shown in NO.7 and SEQ ID NO.8, nucleotide sequence shown in SEQ ID NO.9 and SEQ ID NO.10, SEQ
Nucleotide sequence shown in ID NO.11 and SEQ ID NO.12, nucleotides sequence shown in SEQ ID NO.13 and SEQ ID NO.14
Row, nucleotide sequence shown in SEQ ID NO.15 and SEQ ID NO.16, core shown in SEQ ID NO.17 and SEQ ID NO.18
Nucleotide sequence, nucleotide sequence shown in SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22
Shown nucleotide sequence.
Present invention also offers the application method of the kit, comprise 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 in the kit；
Using the cDNA amplification systems in the kit, the cDNA being prepared with the mRNA primer pairs carries out qRT-
PCR is expanded.
The present invention is during hypoxia adaptability mRNA is detected, described extraction liver and lung tissue sample total serum IgE step
Suddenly extract liver with above-mentioned mRNA screening technique and lung tissue sample total serum IgE step is consistent, will not be repeated here.
The present invention carries out tissue sample cDNA preparation after tissue sample total serum IgE is extracted.Heretofore described tissue sample
Product cDNA preparation 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 synthesis cDNA is carried out to the tissue sample total serum IgE of extraction.Specific reaction includes two steps：The first step removes base
Because of group DNA and second step reverse transcription synthesis cDNA.The first step removes 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×gDNA wiper Mix | 2.0μL |
| Total RNA | 0.5μg |
| Nuclease-free H2O | Add to 8.0 μ L |
The present invention by the first step react needed for mentioned reagent mix after be placed in 42 DEG C reaction 2min.
Second step reverse transcription synthesis cDNA reaction system such as table 2 in the present invention.
The reverse transcription of table 2 synthesizes cDNA reaction system
| Reagent | Usage amount |
| The reaction solution of the first step | 8.0μL |
| 5×HiScript II Q RT SuperMix II | 2.0μL |
| Cumulative volume | 10.0μL |
The present invention by above-mentioned second step reaction component it is well mixed after480 II type quantitative fluorescent PCRs
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.Reverse
After record, the present invention adds 90 μ L Nuclease-free H into obtained reverse transcription system2O is diluted to 100 μ L, obtains
cDNA.In the present invention, the cDNA is standby under the conditions of being stored in -20 DEG C.
The present invention, using the cDNA as template, is expanded after cDNA is obtained with mRNA qRT-PCR primer pairs cDNA
Increase.It is specific in the present invention to use Green PCRKit kits (Qiagen, Germany)
QRT-PCR amplifications are carried out, are existed using the cDNA of above-mentioned acquisition as template480 II type quantitative real time PCR Instruments
Fluorescent quantitative PCR is carried out on (Roche, Swiss).The fluorescent quantitative PCR system is as shown in table 3.
The fluorescent quantitative PCR system of table 3
Quantitative fluorescent PCR program：95℃5min；95 DEG C of 10s, 60 DEG C of 30s, 40 circulations；Circulation utilizes melting after terminating
Curve detection product specificities, 97 DEG C are to slowly warm up to from 60 DEG C, 5 fluorescence signals of every 1 DEG C of collection.
Specific candidate differential expression mRNA expression is calculated according to qRT-PCR relative quantification formula
According to qRT-PCR relative quantification formula：2-ΔΔCt, 11 specific candidate differential expression mRNA are respectively compared 4
Expression water in individual tibetan sheep colony (2 High aititude colonies and 2 intermediate altitude colonies) and control group (low altitude area) sheep colony
It is flat.
Functional annotation, enrichment (GO, KEGG) and egg are carried out by the hypoxia adaptability differential expression mRNA obtained to detection
White interaction network analysis, help to illustrate Adaptive mechanism of the tibetan sheep under low-oxygen environment stress, it is anti-to enrich extreme environment
Basic theory in terms of hay animal molecular ecology adaptability, and provide section for tibetan sheep molecular breeding and functional genomics research
Learn support.
A kind of applications of the mRNA provided by the invention in the detection of tibetan sheep hypoxia adaptability is entered with reference to embodiment
Row detailed description, but they can not be interpreted as limiting the scope of the present invention.
Embodiment 1
The screening of Tibetan plateau sheep liver and lung tissue hypoxia adaptability mRNA
First, material and method
1st, material
Tibetan sheep liver and lung tissue sample information are as follows：
2nd, method
The extraction of 2.1 tibetan sheep livers and lung tissue total serum IgE
According to Life Technologies companiesReagent (article No. 15596026) specification extracts liver respectively
The dirty and Total RNA of lung tissue, then surveyed with the spectrophotometers of NanoDrop 2000 (Thermo Scientific, USA)
Fixed extracted RNA purity and concentration, agarose gel electrophoresis quality inspection ensure the RNA of extraction integrality.
2.2RNA library construction
2.2.1 sample rRNA is removed using epicentre Ribo-ZeroTM kits；
2.2.2 Fragmentation Buffer are added to interrupt rRNA-depleted RNA at random；
2.2.3 using rRNA-depleted RNA as template, synthesized with hexabasic base random primer (random hexamers)
First cDNA chain, then add buffer solution, dATP, dUTP, dCTP, dGTP, RNase H and DNA polymerase I synthesis
Article 2 cDNA chains, cDNA is purified 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 with AMPure XP
Beads carries out clip size selection；
2.2.5 last degraded chain containing U, is enriched with to obtain cDNA library by PCR.
2.3 library Quality Controls
2.3.1 it is tentatively quantitative using Qubit2.0 progress, use Agilent 2100 to carry out the Insert Size in library
Detection, Insert Size can just carry out next step experiment after meeting expection；
2.3.2Q-PCR method carries out accurate quantitative analysis (library valid density ＞ 2nM) to the valid density in library, completes storehouse
Inspection.
Machine is sequenced on 2.4
After storehouse 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.
3rd, data analysis
3.1 sequencing datas and its quality control
Controlled by sequencing quality, obtain 379Gb Clean Data altogether, the Clean Data data obtained are carried out
Data prediction and homogenization, each sample Q30 base percentages reach more than 85%.
3.2 Quality Control data and reference gene group sequence alignment
Using TopHat2 softwares, using genome Oar_v3.1 as reference gene group sequence carry out sequence alignment and after
Continuous mRNA analyses, Oar_v3.1 download address：//ftp.ensembl.org/pub/release-78/fasta/ovis_ aries/。
3.3 transcripts splice
Mapped Reads are subjected to transcript splicing using Cufflinks programs.
3.4 new genes are analyzed
Based on selected reference gene group sequence, Mapped Reads are spliced using Cufflinks softwares, and with original
Some genome annotation information is compared analysis, finds the original transcriptional domain not being annotated, excavates the new transcript of the species
And new gene, so as to supplement and improve original genome annotation information, the new gene function of excavation is annotated.
3.4 differential gene expression screening
Fold differences are more than or equal to 2, and false discovery rate is used as screening criteria less than 0.02, are carried out using DESeq softwares
Differential expression analysis between sample sets, obtain the difference expression gene collection between two biological conditions.
4th, result
4.1 present invention obtain 473766 transcripts using Cufflinks software combinations, divide in liver and lung tissue
Do not excavate new mRNA 2728 and 750, by BLAST softwares by the new mRNA of excavation respectively with NR, Swiss-Prot, GO,
COG, KEGG database carry out sequence alignment, wherein 1153 and 588 mRNA respectively obtain functional annotation.
4.2 present invention relatively Different Altitude tibetan sheep with being total to Recognition Different respectively in control group sheep liver and lung tissue
Express mRNA495 and 233.
4.3 present invention carry out cluster analysis to tibetan sheep liver and lung tissue differential expression mRNA respectively.
4.4 present invention carry out GO enrichments level, GO and COG to tibetan sheep liver and lung tissue differential expression mRNA respectively
Classification, annotation and path enrichment (KEGG) and interactions between protein network analysis.
4.5 present invention are respectively to Different Altitude gradient tibetan sheep and the difference mRNA of control group sheep liver and lung tissue
Analyzed, find 99 (Fig. 1) and 63 (Fig. 2) specific difference expression mRNA respectively in liver and lung tissue.
4.6 present invention are respectively to the difference of same altitudinal gradient tibetan sheep and control group sheep different tissues (liver and lungs)
Different mRNA is analyzed, and in A Wang tibetan sheeps, bar tibetan sheep, the white tibetan sheep in Qilian and Gan Jia tibetan sheeps excavate 101 respectively suddenly
(Fig. 3), 55 (Fig. 4), 56 (Fig. 5) and the individual specific difference expression mRNA of 49 (Fig. 6).
4.7 present invention are respectively compared Different Altitude tibetan sheep with having screened 11 altogether in control group sheep liver and lung tissue
Individual Hypoxia adaptation specific candidate differential expression mRNA.
Embodiment 2
Tibetan plateau sheep liver and lung tissue Hypoxia adaptation specific candidate differential expression mRNA qRT-PCR are examined
Survey
First, material and method
1st, material
With embodiment 1.
2nd, method
2.1 specific candidate differential expression mRNA design of primers
It is right to be preferably used in the primer 11 of dye class qRT-PCR checkings, primer sequence, annealing temperature and product length information
Such as following table：
The extraction of 2.2 tibetan sheeps and control group 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) the total serum IgE reverse transcription to extraction synthesizes cDNA.
The first step removes genomic DNA reaction, and reaction system and condition are as follows：
| Reagent | Usage amount |
| 4×gDNA wiper Mix | 2.0μL |
| Total RNA | 0.5μg |
| Nuclease-free H2O | 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 follows：
| Reagent | Usage amount |
| The reaction solution of the first step | 8.0μL |
| 5×HiScript II Q RT SuperMix II | 2.0μL |
| Cumulative volume | 10.0μL |
By above-mentioned component it is well mixed afterOn 480 II type quantitative real time PCR Instruments (Roche, Swiss)
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 dilutes
To 100 μ L, it is standby to be stored in -20 DEG C of refrigerators.
2.4 dye class fluorescence quantitative PCR detection specific candidate differential expression mRNA expression quantity
Using Green PCR Kit kits (Qiagen, Germany), with reverse transcription
CDNA is that template existsQuantitative fluorescent PCR expansion is carried out on 480 II type quantitative real time PCR Instruments (Roche, Swiss)
Increase.
Dye class quantitative fluorescent PCR system is as follows：
Quantitative fluorescent PCR program：95℃5min；95 DEG C of 10s, 60 DEG C of 30s, 40 circulations；Circulation utilizes melting after terminating
Curve detection product specificities, 97 DEG C are to slowly warm up to from 60 DEG C, 5 fluorescence signals of every DEG C of collection.
2.5 calculate specific candidate differential expression mRNA expression quantity according to qRT-PCR relative quantification formula
According to qRT-PCR relative quantification formula：2-ΔΔCt, 11 specific candidate differential expression mRNA are respectively compared at 4
Expression in tibetan sheep colony (2 High aititude colonies and 2 intermediate altitude colonies) and control group (low altitude area) sheep colony.
As a result it is as shown in Figure 7：QRT-PCR stable amplification results, wherein 5 specific candidate differential expression mRNA
(ENSOARG00000005252、ENSOARG00000007592、ENSOARG00000008263、ENSOARG00000010493、
ENSOARG00000010939) liver organization present expression significantly up-regulation, relative expression levels 1.1215-6.0860 it
Between, hence it is evident that higher than control group (low altitude area) sheep liver organization relative expression levels；6 specific candidate differential expression mRNA
(ENSOARG00000002655、ENSOARG00000008620、ENSOARG00000009196、ENSOARG00000014659、
ENSOARG00000014870, ENSOARG00000014554) present to express with the rise of height above sea level in liver organization and significantly lower,
Relative expression levels are between 0.0060-0.9004, hence it is evident that less than control group (low altitude area) sheep liver organization relative expression's water
It is flat.
Wherein 5 specific candidate differential expression mRNA (ENSOARG00000002655, ENSOARG00000005252,
ENSOARG00000008263, ENSOARG00000010939, ENSOARG00000014554) it is aobvious in lung tissue presentation expression
Up-regulation is write, relative expression levels are between 1.2226-51.6729, hence it is evident that higher than control group (low altitude area) sheep lung tissue phase
To expression；Remaining 6 specific candidate differential expression mRNA (ENSOARG00000007592,
ENSOARG00000008620、ENSOARG00000009196、ENSOARG00000010493、ENSOARG00000014659、
ENSOARG00000014870) lung tissue present expression significantly first lower, relative expression levels 0.1856-0.8803 it
Between, hence it is evident that less than control group (low altitude area) sheep lung tissue relative expression levels.Therefore, the results showed that qRT-PCR results with
RNA-seq results are consistent.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
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 applications of mRNA in the detection of tibetan sheep hypoxia adaptability
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Claims (10)
1. applications of a kind of mRNA in the detection of tibetan sheep hypoxia adaptability, it is characterised in that the mRNA is
ENSOARG00000002655、ENSOARG00000005252、ENSOARG00000007592、ENSOARG00000008263、
ENSOARG00000008620、ENSOARG00000009196、ENSOARG00000010493、ENSOARG00000010939、
ENSOARG00000014554, ENSOARG00000014659 and ENSOARG00000014870.
2. a kind of tibetan sheep hypoxia adaptability correlation mRNA screening technique, comprises the following steps：
1) Different Altitude tibetan sheep and control group sheep liver are extracted respectively and lung tissue sample carries out mRNA sequencings, by dividing
Analysis obtains Clean Data data；
2) the Clean Data data are analyzed and obtains Different Altitude tibetan sheep and control group sheep differential expression mRNA；
3) candidate that significant difference is expressed in Different Altitude tibetan sheep and control group sheep liver and lung tissue sample is screened
MRNA is the hypoxia adaptability mRNA for detecting and obtaining.
3. screening technique according to claim 2, it is characterised in that the Different Altitude tibetan sheep includes High aititude Ba Zang suddenly
The white tibetan sheep of sheep and A Wang tibetan sheeps and intermediate altitude Qilian and Gan Jia tibetan sheeps.
4. screening technique according to claim 2, it is characterised in that the step 1) comprises the following steps：1.1) extraction is hidden
Sheep and the total serum IgE of control group sheep liver and lung tissue sample；
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 qualified cDNA library of quality inspection is sequenced, Clean Data data is obtained by analyzing.
5. according to the screening technique of claim 2 or 4, it is characterised in that the step 2) comprises the following steps：
2.1) the Clean Data data are pre-processed and uniformed, Q30 bases percentage is more than 85%, had
Imitate data；
2.2) valid data that the step 2.1) obtains and reference gene group Oar_v3.1 are subjected to sequence alignment, obtained
Mapped Reads；
2.3) the Mapped Reads are spliced using Cufflinks programs；Carried out with reference gene group annotation information
Comparative analysis, assembling obtain transcript；
2.4) excavated in the transcript and obtain differential expression mRNA.
6. screening technique according to claim 5, it is characterised in that also include after obtaining the differential expression mRNA：To institute
State differential expression mRNA and carry out functional analysis.
7. screening technique according to claim 6, it is characterised in that the functional analysis includes functional annotation, enrichment and egg
White interaction network analysis.
8. screening technique according to claim 7, it is characterised in that it is described be enriched with the database that uses include GO databases and
KEGG databases.
9. a kind of kit for detecting tibetan sheep hypoxia adaptability mRNA, it is characterised in that including following components：Total serum IgE it is anti-
The amplification system of transcription system and cDNA；The reverse transcription system of the total serum IgE includes total serum IgE, reverse transcriptase, dNTP and
oligdT；The amplification system of the cDNA includes mRNA primers：ENSOARG00000002655、
ENSOARG00000005252、ENSOARG00000007592、ENSOARG00000008263、ENSOARG00000008620、
ENSOARG00000009196、ENSOARG00000010493、ENSOARG00000010939、ENSOARG00000014554、
ENSOARG00000014659 and ENSOARG00000014870 primer, above-mentioned mRNA primer sequence are followed successively by SEQ ID
Nucleotide sequence shown in NO.1 and SEQ ID NO.2, nucleotide sequence shown in SEQ ID NO.3 and SEQ ID NO.4, SEQ ID
Nucleotide sequence shown in NO.5 and SEQ ID NO.6, nucleotide sequence shown in SEQ ID NO.7 and SEQ ID NO.8, SEQ ID
Nucleotide sequence shown in NO.9 and SEQ ID NO.10, nucleotide sequence shown in SEQ ID NO.11 and SEQ IDNO.12, SEQ
Nucleotide sequence shown in ID NO.13 and SEQ ID NO.14, nucleotides sequence shown in SEQ ID NO.15 and SEQ ID NO.16
Row, nucleotide sequence shown in SEQ ID NO.17 and SEQ IDNO.18, nucleosides shown in SEQ ID NO.19 and SEQ ID NO.20
Acid sequence, nucleotide sequence shown in SEQ ID NO.21 and SEQ ID NO.22.
10. the application method of kit described in claim 9, it is characterised in that comprise 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, the cDNA being prepared with the mRNA primer pairs carries out qRT-PCR amplifications.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110800690A (en) * | 2019-11-28 | 2020-02-18 | 西藏自治区农牧科学院畜牧兽医研究所 | Method for breeding sheep suitable for high and cold environment through multiple-character selective hybridization |
| CN112863595A (en) * | 2021-03-08 | 2021-05-28 | 中国农业科学院兰州畜牧与兽药研究所 | Method for excavating Tibetan sheep high-altitude hypoxia adaptability related gene based on MeRIP-Seq technology |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002271A (en) * | 2015-07-03 | 2015-10-28 | 西北农林科技大学 | Rapid screening method for sheep hypoxia adaptability and application thereof |
-
2017
- 2017-09-27 CN CN201710885545.9A patent/CN107586855A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002271A (en) * | 2015-07-03 | 2015-10-28 | 西北农林科技大学 | Rapid screening method for sheep hypoxia adaptability and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| WEI CAIHONG等: "Genome-wide analysis reveals adaption to high altitudes in Tibetan sheep", 《NATURE SCIENTIFIC REPORTS》 * |
| 陶海溪等: "哈萨克羊和藏绵羊尾部脂肪组织差异表达基因和可变剪接的鉴定与分析", 《西北农业学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110800690A (en) * | 2019-11-28 | 2020-02-18 | 西藏自治区农牧科学院畜牧兽医研究所 | Method for breeding sheep suitable for high and cold environment through multiple-character selective hybridization |
| CN110800690B (en) * | 2019-11-28 | 2021-12-10 | 西藏自治区农牧科学院畜牧兽医研究所 | Method for breeding sheep suitable for high and cold environment through multiple-character selective hybridization |
| CN112863595A (en) * | 2021-03-08 | 2021-05-28 | 中国农业科学院兰州畜牧与兽药研究所 | Method for excavating Tibetan sheep high-altitude hypoxia adaptability related gene based on MeRIP-Seq technology |
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