CN109112194B - Application of cynoglossus semilaevis sex label piR-mmu-72274 - Google Patents

Application of cynoglossus semilaevis sex label piR-mmu-72274 Download PDF

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CN109112194B
CN109112194B CN201811020134.4A CN201811020134A CN109112194B CN 109112194 B CN109112194 B CN 109112194B CN 201811020134 A CN201811020134 A CN 201811020134A CN 109112194 B CN109112194 B CN 109112194B
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CN109112194A (en
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张博
贾磊
高磊
赵娜
刘克奉
鲍宝龙
李仰真
车金远
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TIANJIN BOHAI SEA FISHERIES RESEARCH INSTITUTE
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Abstract

The invention relates to an application of cynoglossus semilaevis gender label piR-mmu-72274, wherein gender label piR-mmu-72274 is derived from fish seminal plasma exosomes, labeled piRNAs which are obviously and differentially expressed in two types of fish are screened as candidates through small RNA sequencing analysis, and a piRNA biomarker which has an indicating effect on the two types of fish is finally determined through real-time quantitative PCR verification, wherein the labeled piRNAs are piR-mmu-72274, the sequence is GCATTGGTGGTTCAGTGGTAGAATTCTCGCCT, and a detection kit is developed and prepared on the basis of the labeled piRNAs. The method has the advantages of no wound, high efficiency and reliable identification result, and is used for judging the genetic sex of the cynoglossus semilaevis by quantitative detection for the first time.

Description

Application of cynoglossus semilaevis sex label piR-mmu-72274
Technical Field
The invention belongs to the technical field of fish biology, and particularly relates to application of a cynoglossus semilaevis gender label piR-mmu-72274.
Background
The Cynoglossus semilaevis (Cynoglossus semilaevis) is restricted by certain degree in industrial development due to the great difference of the growth speed and body type of female and male fish, high proportion of male fish in cultivation and low economic value. Studies have found that a significant proportion of male fish are pseudomale, i.e. genetically female, but phenotypically male. The pseudo-male fish is used as a male parent, and the offspring can inherit the characteristics of the father to become the pseudo-male fish, so that the problem of high male fish ratio is caused. Therefore, the identification of the pseudo-male fish has important significance for the breeding industry of the cynoglossus semilaevis. The traditional female specific microsatellite molecular marker method needs agarose electrophoresis band identification and has certain error; the accuracy of chromosome identification is limited by the effect of the control chip, and both methods are qualitative judgments. The invention develops the cynoglossus semilaevis seminal plasma exosome piRNAs marker, quantitatively inspects the expression quantity of piRNA by applying a quantitative PCR method to realize the identification of male cynoglossus semilaevis and pseudo-male fish, and has the advantages of high accuracy and stable and reliable result.
PiRNA (Piwi-interacting RNA) which is mainly present in the reproductive system of animals is a type of non-coding RNA of about 26-32nt (nucleotides) in length. piRNA plays an important role in animal development and reproductive regulation, and in drosophila ovary germ cells, the piRNA is greatly amplified in the cells through Ping-Pong circulation (Ping-Pong cycle) through the coordination and processing of PIWI family proteins, and a specific action mechanism of the piRNA is yet to be discovered. The research of the source of exosome piRNAs as biomarkers in marine animals is hardly reported, and the invention develops the plasma exosome piRNAs marker of the cynoglossus semilaevis, solves the problem of identification of the genetic sex of the male cynoglossus semilaevis and the pseudo-male fish, and has innovation.
Disclosure of Invention
The invention aims to solve the technical problem of providing an application of a cynoglossus semilaevis sex label piR-mmu-72274, wherein the sex label piR-mmu-72274 is derived from seminal plasma exosome piRNA of the cynoglossus semilaevis so as to solve the problem of distinguishing the genetic sex of male and pseudo-male cynoglossus semilaevis. The research of the source of exosome piRNA as a biomarker in marine animals is hardly reported, and the invention develops a method for extracting and primarily applying exosome from the source of seminal plasma represented by cynoglossus semilaevis, and solves the problem of identification of the genetic sex of the male cynoglossus semilaevis and the pseudo-male fish.
The technical problem to be solved by the invention is realized by adopting the following technical scheme:
an application of a cynoglossus semilaevis sex label piR-mmu-72274 is disclosed, wherein the sex label piR-mmu-72274 is derived from plasma exosome piRNA of cynoglossus semilaevis, and the sequence is GCATTGGTGGTTCAGTGGTAGAATTCTCGCCT.
The invention also provides a screening method of the seminal plasma exosome piR-mmu-72274, which comprises the following specific steps:
1) after the seminal plasma exosomes are separated and identified, sequencing analysis is carried out on the seminal plasma exosome smallrnas, Small RNAs are various, clean reads are sequentially compared with an Rfam database, a cDNA sequence, a species repetitive sequence library and a piRBase database, so that the smallrnas in a sequencing result are classified and annotated; comparing the annotated sequence with a piRBase database, and carrying out statistics on known piRNA; carrying out new piRNA prediction, piRNA differential expression analysis, pathway enrichment analysis and function enrichment analysis of differential expression piRNA target genes by using the unannotated sequences, finally carrying out correlation matching with male fish and pseudo-male fish samples, and screening out one or more piRNAs which have indication effects on the male fish and the pseudo-male fish samples and serve as candidate biomarkers;
2) two validation samples were taken: quantitatively detecting the differential expression condition of candidate piRNA by using a MicroRNA quantitative analysis kit for the total RNA of the sperms of the male fish and the pseudo-male fish identified by the peripheral blood chromosome, and screening the piRNA with the most label indication function as the final label piRNA; according to the statistical analysis principle, the P value less than 0.05 is a significant difference; the piRNA biomarker piR-mmu-72274 with sequence GCATTGGTGGTTCAGTGGTAGAATTCTCGCCT was screened for differential expression in males and pseudomales.
The invention provides a detection kit for male cynoglossus semilaevis and pseudo-male fish, which is characterized in that: the kit comprises piR-mmu-72274 quantitative detection primers, wherein the primer sequences are F: GCATTGGTGGTTCAGTGGT, R: TCGTATCCAGTGCAGGGTC, a reverse transcription primer piR-mmu-72274-RT: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAAGGCGAGA, and other PCR reverse transcription reagents and PCR fluorescent quantitative reagents, the internal reference piR is U6.
Further, the U6 primer is U6-F: CTCGCTTCGGCAGCACATATACT, respectively; U6-R: ACGCTTCACGAATTTGCGTGTC are provided.
Furthermore, the 5 'end of the primer probe sequence is marked with a fluorescent group, and the 3' end is marked with a quenching group, so that the detection is carried out by a method suitable for Taqman probe-based qRT-PCR.
Compared with the prior art, the invention has the beneficial effects
The invention innovatively applies the semen exosome piRNA to carry out the sex identification of the cynoglossus semilaevis, develops a detection kit, belongs to the first time in aquatic animals, has the advantages of no wound and high efficiency, has reliable identification result, and is used for distinguishing the genetic sex of the cynoglossus semilaevis by quantitative detection for the first time.
Drawings
FIG. 1 shows qRT-PCR expression of the marker piRNA of the present invention after 20 validation samples;
FIG. 2 mean values of qRT-PCR expression in two groups of samples after validation of 20 validation samples according to the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments, which are illustrative only and not intended to be limiting, and the scope of the present invention is not limited thereby.
Example 1
A screening method of cynoglossus semilaevis semen-derived exosome piR-mmu-72274 as a biomarker.
1. Semen sample collection
Collecting 0.5ml of sperm of male fish of sexual maturity of cynoglossus semilaevis in a centrifuge tube for exosome separation and identification.
2. Exosome extraction
(1) Transferring 0.5ml semen sample to 1.5ml EP tube, placing at 4 deg.C, centrifuging at 1200g for 15min to remove sperm cells, centrifuging at 4 deg.C at 15000g for 20min to remove small cell impurities and debris, diluting with PBS 1 times, pre-filtering with 0.45um filter membrane, and filtering the filtrate with 0.22um filter membrane.
(2) The filtered sample was used to extract exosomes using Total Exosome Isolation Kit.
(3) The lysates were collected and transferred completely to a rnase-free 2ml tube.
3. Identification of exosomes: and (4) observing by using a Hitachi H600IV transmission electron microscope, and analyzing and identifying by using the transmission electron microscope, wherein the rest sample is used for RNA extraction and sequencing analysis.
4. PiRNA sequencing analysis of exosomes
And (3) extracting RNA of the seminal plasma exosomes by a TRizol method, and sequencing and analyzing the smallRNA of the seminal plasma exosomes. Small RNAs are various in types, including miRNA, tRNA (tirRNA, tRNAs), rRNA, piRNA, snorRNA and the like, a Small RNA library is constructed after quality inspection, sequencing is carried out based on an illumina platform, data filtering analysis is carried out after sequencing is completed, and clean reads are sequentially compared and annotated with an Rfam database, a cDNA sequence, a species repetitive sequence library and a piRBase database. The filtered sequences were aligned with the piRBase database and statistics of known pirnas were performed. And performing new piRNA prediction, piRNA differential expression analysis, pathway enrichment analysis and function enrichment analysis of the target gene of the differentially expressed piRNA by using the unannotated sequence, and finally performing correlation matching with two samples, namely a male fish sample and a pseudo-male fish sample, and screening out one or more piRNAs which have indication effects on the two samples of the male fish and the pseudo-male fish as candidate biomarkers.
5. Real-time fluorescent quantitative PCR analysis verification
Extracting the total RNA of exosomes from the seminal plasma of the male fish and the pseudo-male fish, quantitatively detecting the expression condition of the piRNA in two samples to be detected by utilizing a MicroRNA quantitative analysis kit, and selecting 2 from U6 which has stable and high expression in two groups of samples by internal reference-ΔΔCtAnd performing calculation analysis. The piRNA biomarker piR-mmu-72274 with sequence GCATTGGTGGTTCAGTGGTAGAATTCTCGCCT was screened for differential expression in males and pseudomales.
Example 2
Kit for identifying male fish and pseudo-male fish by cynoglossus semilaevis semen exosome miRNA marker
The identification kit based on the semen exosome piRNA marker piR-mmu-72274 comprises piR-mmu-72274 quantitative detection primers, wherein the primer sequences are F: GCATTGGTGGTTCAGTGGT, R: TCGTATCCAGTGCAGGGTC, PCR reverse transcription reagent and PCR fluorescent quantitative reagent, the internal reference piR is U6, and the primers are U6-F: CTCGCTTCGGCAGCACATATACT, respectively; U6-R: ACGCTTCACGAATTTGCGTGTC, respectively; the primers comprise reverse transcription primer piR-mmu-72274-RT: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAAGGCGAGA, the kit additionally contains other conventional reagents for quantitative PCR reactions: reverse transcriptase, Taq enzyme, dNTP, buffer, Mgcl2DEPC water and control. The reaction system applied by the kit is a 10ul system, namely 0.5ul 10 x miRNA primer probe, 5ul 2 x Master mix and 2.5ul ddH2O, 2ul cDNA template. Real-time fluorescent quantitative PCR detection using Q6 from Thermo fisherThe procedure should be: 2min at 95 ℃; the temperature is 95 ℃ for 10s, the temperature is 59 ℃ for 60s, and the circulation is carried out for 40 times. The samples were tested in 3 replicates. The expression conditions of the labeled piRNAs of the chromosome-confirmed 10-cynoglossus semilaevis pseudo-male fish and 10-cynoglossus semilaevis male fish, namely piR-mmu-72274, are detected by using the screened piR-mmu-72274 quantitative detection primer and the reverse transcription primer, and the results are shown in Table 1, FIG. 1 and FIG. 2. The expression difference of the label in two groups of samples is very obvious (p is less than 0.01), and the detection accuracy rate of the label piRNA is verified to be more than 90%.
TABLE 120 expression of Cynoglossus semilaevis validation tag piR-mmu-72274
Figure BDA0001787094480000051
Figure BDA0001787094480000061
Figure BDA0001787094480000071
For those skilled in the art, the conception and the technical point of the present invention may be changed, and the corresponding changes should be covered by the claims of the present invention.
Sequence listing
<110> Tianjin Bohai sea aquatic product research institute
Application of sex label piR-mmu-72274 of cynoglossus semilaevis
<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> 32
<212> DNA
<213> Cynoglossus semilaevis Gunther (Cynoglossus semilaevis Gunther)
<400> 3
gcattggtgg ttcagtggta gaattctcgc ct 32
<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 cgaaggcgag a 51

Claims (3)

1. An application of a cynoglossus semilaevis sex label piR-mmu-72274 in cynoglossus semilaevis sex identification is disclosed, wherein the sex label piR-mmu-72274 is derived from plasma exosome piRNA of cynoglossus semilaevis, and the sequence is GCATTGGTGGTTCAGTGGTAGAATTCTCGCCT.
2. The application of cynoglossus semilaevis sex label piR-mmu-72274 in cynoglossus semilaevis sex identification according to claim 1, wherein the screening method of seminal plasma exosome piRNA comprises the following steps:
1) after the seminal plasma exosomes are separated and identified, sequencing analysis is carried out on the seminal plasma exosome smallrnas, Small RNAs are various, clean reads are sequentially compared with an Rfam database, a cDNA sequence, a species repetitive sequence library and a piRBase database, so that the smallrnas in a sequencing result are classified and annotated; comparing the annotated sequence with a piRBase database, and carrying out statistics on known piRNA; carrying out new piRNA prediction, piRNA differential expression analysis, pathway enrichment analysis and function enrichment analysis of differential expression piRNA target genes by using the unannotated sequences, finally carrying out correlation matching with male fish and pseudo-male fish samples, and screening out one or more piRNAs which have indication effects on the male fish and the pseudo-male fish samples and serve as candidate biomarkers;
2) two random sets of validation samples were extracted: quantitatively detecting the differential expression condition of candidate piRNA by using a MicroRNA quantitative analysis kit for the total RNA of the sperms of the male fish and the pseudo-male fish identified by the peripheral blood chromosome, and screening the piRNA with the most label indication function as the final label piRNA; according to the statistical analysis principle, the P value less than 0.05 is a significant difference; the piRNA biomarker piR-mmu-72274 with sequence GCATTGGTGGTTCAGTGGTAGAATTCTCGCCT was screened for differential expression in males and pseudomales.
3. The utility model provides a cynoglossus semilaevis male fish and pseudo-male fish detect reagent box which characterized in that: the kit comprises a sex label piR-mmu-72274 quantitative detection primer, wherein the sequence of the primer is F: GCATTGGTGGTTCAGTGGT, and the sequence of a R: TCGTATCCAGTGCAGGGTC, PCR reverse transcription primer is piR-mmu-72274-RT: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAAGGCGAGA, and other reverse transcription reagents and qPCR fluorescent quantitation reagents, the internal reference piR is U6.
CN201811020134.4A 2018-09-03 2018-09-03 Application of cynoglossus semilaevis sex label piR-mmu-72274 Expired - Fee Related CN109112194B (en)

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CN101225437A (en) * 2007-12-01 2008-07-23 中国水产科学研究院黄海水产研究所 Tongue sole molecular marker auxiliary sex control method

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CN101225437A (en) * 2007-12-01 2008-07-23 中国水产科学研究院黄海水产研究所 Tongue sole molecular marker auxiliary sex control method

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Title
Identification and application of piwiinteracting RNAs from seminal plasma exosomes in Cynoglossus semilaevis;Bo Zhang等;《BMC Genomics》;20200415;1-11 *
半滑舌鳎养殖群体的性比与雌雄形态差异比较;李虎等;《水产学报》;20120930;第36卷(第9期);1331-1336 *

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