CN109971863A - The application of Cynoglossus semilaevis gender label piR-mmu-29271668 - Google Patents
The application of Cynoglossus semilaevis gender label piR-mmu-29271668 Download PDFInfo
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- 241001014350 Cynoglossus semilaevis Species 0.000 title claims abstract description 34
- 230000029142 excretion Effects 0.000 claims abstract description 29
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- 238000001514 detection method Methods 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 12
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- 229920000936 Agarose Polymers 0.000 description 1
- 241000269335 Ambystoma laterale x Ambystoma jeffersonianum Species 0.000 description 1
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
A kind of application of Cynoglossus semilaevis gender label piR-mmu-29271668, the gender label piR-mmu-29271668 starches excretion body from milt, the present invention passes through small RNA sequencing analysis, the label piRNAs of two kinds of fish significant difference expression is screened as candidate, it is verified by real-time quantitative PCR, finally determine to two kinds of fishes piRNA biomarker with indicating function, label piRNAs is piR-mmu-29271668, sequence is GCATTGGTGGTTCAGTGGTAGAATTTTGGA, and exploitation is prepared for detection kit based on this.The method of the present invention has noninvasive, efficient advantage, and qualification result is reliable, is first passage quantitative detection to differentiate the genetic sex of Cynoglossus semilaevis.
Description
Technical field
The invention belongs to fish biotechnology fields, more particularly to a kind of Cynoglossus semilaevis gender label piR-mmu-
29271668 application.
Background technique
Cynoglossus semilaevis (Cynoglossus semilaevis) due to female the milter speed of growth and figure greatest differences,
And milter ratio is high in cultivation, the low problem of economic value, industry development is by a degree of restriction.Research finds milter
In have the pseudo-milter of significant proportion, i.e., be genetically raun, but be milter in phenotype.And use pseudo-milter as male parent, Hou Daiye
The feature of the hereditary father of meeting becomes pseudo-milter, results in the high problem of milter ratio.Therefore identify pseudo-milter for Cynoglossus semilaevis
Breeding industries have great importance.The special microsatellite molecular marker method of traditional female needs agarose electrophoresis band to identify,
With certain error；And Chromosome Identification accuracy is restricted by production effect, both methods is all qualitatively to judge.This
Invention develops Cynoglossus semilaevis refining excretion body piRNAs marker, using the expression of the method quantitative expedition piRNA of quantitative PCR
Amount identifies Cynoglossus semilaevis milter and pseudo-milter to realize, the advantage high with accuracy, result is reliable and stable.
It is about 26- that the piRNA (Piwi-interactiing RNA) for being primarily present in animals' reproduction system, which is a kind of length,
The non-coding RNA of 32nt (nucleotides).PiRNA plays an important role in animal development and genital regulating, in drosophila ovary
In reproduction cell, collaboration, processing through PIWI family protein, piRNA is by table tennis circulation (" Ping-Pong " cycle) thin
Massive amplification in born of the same parents, specific mechanism of action need to be excavated.The piRNAs in excretion body source is as life in marine animal
The research of object marker is almost without report, a kind of Cynoglossus semilaevis refining excretion body piRNAs marker that the present invention develops,
It solves the problems, such as the identification of Cynoglossus semilaevis milter and pseudo-milter genetic sex, there is novelty.
Summary of the invention
The technical problem to be solved in the present invention is that providing a kind of Cynoglossus semilaevis gender label piR-mmu-29271668's
Using the gender label piR-mmu-29271668 derives from the refining excretion body piRNA of Cynoglossus semilaevis, sliding to solve half
The differentiation problem of tongue sole milter and pseudo-milter genetic sex.The piRNA in excretion body source is as biological marker in marine animal
Almost without report, the present invention is developed to be extracted for the excretion body for starching source as the milt of representative using Cynoglossus semilaevis for the research of object
And the method for Preliminary Applications, solve the problems, such as the identification of Cynoglossus semilaevis milter and pseudo-milter genetic sex.
The present invention solves its technical problem and adopts the following technical solutions to achieve:
A kind of application of Cynoglossus semilaevis gender label piR-mmu-29271668, the gender label piR-mmu-
29271668 derive from refining excretion the body piRNA, sequence GCATTGGTGGTTCAGTGGTAGAATTTTGGA of Cynoglossus semilaevis.
The present invention also provides the screening techniques of refining excretion body piR-mmu-29271668 a kind of, and specific steps are such as
Under:
1) refining excretion body is separated and identified, and carries out sequencing analysis, Small RNA type to refining excretion body smallRNA
It is various, clean reads is successively carried out with Rfam database, cDNA sequence, species repetitive sequence library, piRBase database
It compares, to carry out classification annotation to the smallRNA in sequencing result；By after annotation sequence and piRBase database compare,
Carry out the statistics of known piRNA；It carries out new piRNA using the sequence not annotated to predict, piRNA Differential expression analysis is poor
The access enrichment analysis and function enrichment analysis of different expression piRNA target gene, finally carry out correlation with milter, pseudo-milter sample
Matching filters out one or more kinds of lifes to two kinds of sample piRNA with indicating function of milter and pseudo-milter as candidate
Substance markers object；
2) extract two groups of verifying samples: the milter and pseudo-milter refining source excretion body after peripheral blood chromosome is identified are total
RNA filters out most label using the differential expression situation of MicroRNA quantification assay kit quantitative detection candidate piRNA
The piRNA of indicative function is as final label piRNA；According to statistical analysis principle, P value is significant difference less than 0.05；
The piRNA biomarker piR-mmu-29271668 of the differential expression in milter and pseudo-milter is screened, sequence is
GCATTGGTGGTTCAGTGGTAGAATTTTGGA。
The present invention provides a kind of Cynoglossus semilaevis milter and pseudo-milter detection kits, it is characterised in that: the kit
Including piR-mmu-29271668 quantitative detection primer, primer sequence F:GCATTGGTGGTTCAGTGGT；R:
TCGTATCCAGTGCAGGGTC, reverse transcriptase primer: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGATC
CAAAAT；And PCR reverse transcription reagents and PCR fluorescent quantitation reagent, internal reference piR are U6.
The primer of U6 is U6-F:CTCGCTTCGGCAGCACATATACT；U6-R:ACGCTTCACGAATTTGCGTGTC.
Further, fluorophor, 3 ' end label quenching groups, to be suitble to Taqman are marked in 5 ' ends of primer probe sequence
The detection of probe-based qRT-PCR method.
The beneficial effect of the present invention compared with prior art
Novelty of the invention carries out Cynoglossus semilaevis sex identification with sperm excretion body piRNA, develops the examination of detection
Agent box, still belongs to the first time in aquatic animal, this identification method has noninvasive, efficient advantage, and qualification result is reliable, is for the first time
The genetic sex of Cynoglossus semilaevis is differentiated by quantitative detection.
Detailed description of the invention
Fig. 1 is qRT-PCR expression quantity of the mark piRNA proposed by the present invention after 20 are verified sample verifying；
QRT-PCR expression quantity mean value after 20 verifying sample verifyings of Fig. 2 present invention in two groups of samples.
Specific embodiment
The present invention is further described below in conjunction with specific embodiment, following embodiment be it is descriptive, be not
Limited, this does not limit the scope of protection of the present invention.
Embodiment 1
A kind of screening technique of the excretion body piR-mmu-29271668 in Cynoglossus semilaevis sperm source as biomarker.
1, the collection of semen sample
Cynoglossus semilaevis sexal maturity milter sperm 0.5ml is collected in centrifuge tube for the separation identification of excretion body.
2, the extraction of excretion body
(1) 0.5ml semen sample transfer 1.5mlEP pipe is taken, is placed in 4 DEG C, 1200g is centrifuged 15min and removes spermatoblast,
4 DEG C, 15000g is centrifuged 20min removal cellule impurity and fragment, after diluting 1 times with PBS, carries out pre- mistake using 0.45um filter membrane
Filter, filtered fluid is again through 0.22um membrane filtration.
(2) filtered sample extracts excretion body with Total Exosome Isolation Kit kit.
(3) it collects dissolution sample and is transferred in the 2ml pipe without RNA enzyme completely.
3, the identification of excretion body: Hitachi H600IV type transmission electron microscope observing, after transmission electron microscope analysis identification, remaining sample is used
In RNA extraction and sequencing analysis.
4, the piRNA sequencing analysis of excretion body
TRizol method extracts the RNA of refining excretion body, carries out sequencing analysis to the smallRNA of refining excretion body.Small
RNA is many kinds of, including miRNA, tRNA (tiRNA, tRFs), rRNA, piRNA, snoRNA etc., and tiny RNA text is constructed after quality inspection
Library simultaneously based on illumina platform be sequenced, complete sequencing after carry out data filter analysis, by clean reads successively with Rfam number
Annotation is compared according to library, cDNA sequence, species repetitive sequence library, piRBase database.By filtered sequence and
PiRBase database compares, and carries out the statistics of known piRNA.New piRNA is carried out using the sequence not annotated to predict,
PiRNA Differential expression analysis, differential expression piRNA target gene access enrichment analysis and function enrichment analysis, finally with milter,
Two samples of pseudo-milter carry out relevant matches, filter out one or more, have to two kinds of samples of milter and pseudo-milter and refer to
The piRNA used is shown as candidate biomarker.
5, real-time fluorescence quantitative PCR analysis verifying
Milter and pseudo-milter refining source excretion body total serum IgE are extracted, is quantitatively examined using MicroRNA quantification assay kit
The expression of piRNA in two samples to be detected is surveyed, internal reference selection all has in two groups of samples stablizes highly expressed U6 choosing
Take 2-ΔΔCtCarry out calculating analysis.Screen the piRNA biomarker piR-mmu- of the differential expression in milter and pseudo-milter
29271668, sequence GCATTGGTGGTTCAGTGGTAGAATTTTGGA.
Embodiment 2
The kit of Cynoglossus semilaevis sperm excretion body miRNA marker identification milter and pseudo-milter
Identification kit based on sperm excretion body piRNA marker piR-mmu-29271668 includes piR-mmu-
29271668 quantitative detection primers, primer sequence F:GCATTGGTGGTTCAGTGGT；R:TCGTATCCAGTGCAGGGTC,
PCR reverse transcription reagents and PCR fluorescent quantitation reagent, internal reference piR are U6, primer U6-F:
CTCGCTTCGGCAGCACATATACT；U6-R:ACGCTTCACGAATTTGCGTGTC；Reverse transcriptase primer GTCGTATCCAGTGC
AGGGTCCGAGGTATTCGCACTGGATACGATCCAAAAT, furthermore kit includes other conventional examinations of quantitative PCR reaction
Agent: reverse transcriptase, Taq enzyme, dNTP, buffer, Mgcl2, DEPC water and reference substance.The kit application reaction system be
10ul system, i.e. 0.5ul 10*miRNA primed probe, 5ul 2*Master mix, 2.5ul ddH2O, 2ulcDNA template.Make
It is detected with the Q6 real-time fluorescence quantitative PCR of Thermo fisher, the program of reaction are as follows: 95 DEG C of 2min；95℃10s,59℃60s,
Circulation 40 times.Sample detection sets 3 in parallel.Utilize the quantitative detection primer and reverse transcription of the piR-mmu-29271668 screened
The label piRNA, i.e. piR- of 10 tail Cynoglossus semilaevis pseudo-milters and 10 tail Cynoglossus semilaevis milters that primer detection confirms through chromosome
The expression of mmu-29271668, as a result as shown in table 1, Fig. 1, Fig. 2.Differential expression of the label in two groups of samples is extremely
Significantly (p < 0.01) verifies the Detection accuracy of label piRNA up to 90% or more.
1 20 tail Cynoglossus semilaevis of table verifies the expression of the label piR-mmu-29271668 of sample
For those skilled in the art, the design of the invention being directed to and technical essential are deformed, it is corresponding to change
It should belong to the requested claim of the present invention.
Sequence table
<110>Tianjin Bohai Sea aquatic products research institute
<120>application of Cynoglossus semilaevis gender label piR-mmu-29271668
<160> 6
<170> SIPOSequenceListing 1.0
<210> 5
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
ctcgcttcgg cagcacatat act 23
<210> 6
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
acgcttcacg aatttgcgtg tc 22
<210> 3
<211> 30
<212> DNA
<213>Cynoglossus semilaevis (Cynoglossus semilaevis Gunther)
<400> 3
gcattggtgg ttcagtggta gaattttgga 30
<210> 4
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gcattggtgg ttcagtggt 19
<210> 5
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
tcgtatccag tgcagggtc 19
<210> 6
<211> 51
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 6
gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgatccaaaa t 51
Claims (3)
1. a kind of application of Cynoglossus semilaevis gender label piR-mmu-29271668, the gender label piR-mmu-
29271668 derive from refining excretion the body piRNA, sequence GCATTGGTGGTTCAGTGGTAGAATTTTGGA of Cynoglossus semilaevis.
2. the application of Cynoglossus semilaevis gender label piR-mmu-29271668 according to claim 1 a kind of, feature exist
In the screening technique of the refining excretion body piRNA, the specific steps are as follows:
1) refining excretion body is separated and identified, and carries out sequencing analysis to refining excretion body smallRNA, and SmallRNA is many kinds of,
Clean reads is successively compared with Rfam database, cDNA sequence, species repetitive sequence library, piRBase database,
To carry out classification annotation to the smallRNA in sequencing result；By the sequence and the comparison of piRBase database after annotation, carry out
Know the statistics of piRNA；It carries out new piRNA using the sequence not annotated to predict, piRNA Differential expression analysis, differential expression
The access enrichment analysis and function enrichment analysis of piRNA target gene, finally carry out relevant matches with milter, pseudo-milter sample,
Filter out one or more kinds of biomarkers to two kinds of sample piRNA with indicating function of milter and pseudo-milter as candidate
Object；
2) extract random two groups of verifying samples: the milter and pseudo-milter refining source excretion body after peripheral blood chromosome is identified are total
RNA filters out most label using the differential expression situation of MicroRNA quantification assay kit quantitative detection candidate piRNA
The piRNA of indicative function is as final label piRNA；According to statistical analysis principle, P value is significant difference less than 0.05；
The piRNA biomarker piR-mmu-29271668 of the differential expression in milter and pseudo-milter is screened, sequence is
GCATTGGTGGTTCAGTGGTAGAATTTTGGA。
3. a kind of Cynoglossus semilaevis milter and pseudo-milter detection kit, it is characterised in that: the inclusive distinguishing label of kit
PiR-mmu-29271668 quantitative detection primer, primer sequence F:GCATTGGTGGTTCAGTGGT；R:
TCGTATCCAGTGCAGGGTC, PCR reverse transcriptase primer sequence piR-mmu-29271668-RT:GTCGTATCCAGTGCAGGGT
CCGAGGTATTCGCACTGGATACGATCCAAAAT and other reverse transcription reagents and qPCR fluorescent quantitation reagent, internal reference piR
For U6.
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| CN2018110295657 | 2018-09-03 | ||
| CN201811029565 | 2018-09-03 |
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| PIRBASE: ""Detailed information of piRNA piR-mmu-29271668", 《PIRBASE》 * |
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