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Application of Cynoglossus semilaevis sex tag piR-xtr-979116

A technology of pir-xtr-979116, semi-smooth tongue sole, applied in the application field of semi-smooth tongue sole sex label piR-xtr-979116, to achieve the effect of reliable identification results

Active Publication Date: 2018-12-14
TIANJIN BOHAI SEA FISHERIES RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In marine animals, there is almost no report on the research on exosome-derived piRNA as a biomarker. The present invention develops a method for the extraction and preliminary application of exosomes derived from fish seminal plasma represented by semi-smooth tongue sole, and solves the problem of semi-smooth tongue sole male fish and Discrimination of the Genetic Sex of Pseudo-Male Fish

Method used

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  • Application of Cynoglossus semilaevis sex tag piR-xtr-979116

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Experimental program
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Effect test

Embodiment 1

[0019] A screening method for exosomal piRNA derived from semismooth tongue sole semen as a biomarker

[0020] 1. Collection of semen samples

[0021] Collect 0.5 ml of semen from sexually mature males of semi-smooth tongue sole in a centrifuge tube for the isolation and identification of exosomes.

[0022] 2. Extraction of exosomes

[0023] (1) Transfer 0.5ml semen sample to 1.5ml EP tube, place at 4°C, centrifuge at 1200g for 15min to remove sperm cells, centrifuge at 15000g at 4°C for 20min to remove small cell impurities and debris, dilute 1 times with PBS, and use 0.45um filter membrane Perform pre-filtration, and then filter the filtrate through a 0.22um membrane filter.

[0024] (2) Exosomes were extracted from the filtered samples using the Total Exosome Isolation Kit.

[0025] (3) Collect the lysate and transfer completely to an RNase-free 2ml tube.

[0026] 3. Identification of exosomes: Hitachi H600IV transmission electron microscope observation, after transmiss...

Embodiment 2

[0032] Kit for identification of male and pseudo-male by exosome miRNA markers in semismooth tongue sole semen

[0033] The identification kit based on semen exosome piRNA marker piR-xtr-979116 includes piR-xtr-979116 quantitative detection primers, the primer sequence is, F:TTAATCGGGTTCGTTTCCC, R:TCGTATCCAGTGCAGGGTC, PCR reverse transcription primer piR-xtr-979116-RT : GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGATGGTGCGT and reagents and qPCR fluorescence quantitative reagents, the internal reference piR is U6, the primer is U6-F: CTCGCTTCGGCAGCACATATACT; U6-R: ACGCTTCACGAATTTGCGTGTC; the kit primers include PCR reverse transcription primers, quantitative PCR forward primers and reverse primers, and other reagents The box contains other conventional reagents for quantitative PCR reactions: reverse transcriptase, Taq enzyme, dNTP, buffer, Mgcl 2, DEPC water and reference substance. The reaction system used in this kit is a 10ul system, that is, 0.5ul 10*miRNA primer probe, 5ul...

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Abstract

The invention relates to application of a Cynoglossus semilaevis sex tag that is piR-xtr-979116. The sex tag piR-xtr-979116 is derived from a fish seminal plasma exosome. Through small RNA sequencinganalysis, a tag piRNAs significantly different in expression in two types of fish is obtained through screening and adopted as a candidate, a piRNA biomarker having an indicative function for the twotypes of fish is finally determined through verification adopting real-time quantitative PCR, the tag piRNAs is piR-xtr-979116 with a sequence of CGGGTTCGTTTCCCGGCCAACGCACCA, and a kit is developed based on the tag. A method provided by the invention is noninvasive and efficient, an identification result is reliable and it is the first time to determine the genetic sex of Cynoglossus semilaevis through quantitative determination.

Description

technical field [0001] The invention belongs to the field of fish biotechnology, and in particular relates to the application of a sex tag piR-xtr-979116 in tongue sole. Background technique [0002] piRNA (Piwi-interactiing RNA) is a kind of non-coding RNA with a length of about 26-32nt (nucleotides). It was first discovered in 2006 by four independent research groups in the germline cells of Drosophila, mouse, rat and human. Find. Among all non-coding RNAs, piRNAs are the most numerous and mainly exist in the reproductive system. piRNAs serve as guides for PIWI, regulating the expression of target genes as well as modification at the transcriptional and post-transcriptional levels. piRNAs are different from miRNAs in their sequence characteristics and production methods. They are transcribed and processed from genome sequences and do not require Dicer cutting. Piwi binds to piRNA and enters the nucleus to participate in epigenetic regulation. There is evidence that it m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6879C12Q1/6888
CPCC12Q1/6879C12Q1/6888C12Q2600/178
Inventor 张博贾磊赵娜李坤明鲍宝龙刘克奉李仰真车金远
Owner TIANJIN BOHAI SEA FISHERIES RES INST
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