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Acipenser dabryanus gonad differential expression gene GSDF and screening method thereof

A technology for differentially expressed genes and screening methods, applied in biochemical equipment and methods, recombinant DNA technology, DNA/RNA fragments, etc., which can solve problems such as wasting time and affecting accuracy

Inactive Publication Date: 2018-04-20
FISHERIES INST SICHUAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Generally speaking, the sex-related information of sturgeon is usually dissected, and the gonads are taken out for observation or tissue section identification, which is a waste of time and its accuracy is affected by individual size and gonad development stage

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Dabry's sturgeon sample collection and RNA extraction

[0024] Two healthy Dabry's sturgeons with differentiated gonads were selected from Sichuan Fisheries Research Institute, and their gonads were dissected to extract total RNA. In a bowl, use liquid to quickly grind into powder, put it into a 1.5mL centrifuge tube, and add 1mL TRIzol; (2) Add 200μL of chloroform to the centrifuge tube, shake vigorously for 15s, and stand at room temperature for 3min; (3) 4°C, 12,000r / Centrifuge for 10 min, absorb the supernatant and put it into a new centrifuge tube; (4) Add isopropanol equal to the volume of the supernatant, shake gently to mix, and place at room temperature for 5-10 min; (5) 4°C, 12,000r / min Centrifuge for 10 minutes, discard the supernatant, and carefully absorb the remaining isopropanol; (6) Add 1 mL of 75% ethanol to wash the precipitate thoroughly, centrifuge at 7,500 r / min for 1 minute at 4°C, briefly centrifuge, absorb the remaining ethanol, repeat...

Embodiment 2

[0025] Example 2 Sequencing, splicing and assembly of the transcriptome of Dabry's sturgeon

[0026] After the sample is qualified, the library construction is carried out. The main process is as follows: (1) enrich eukaryotic mRNA with magnetic beads containing Oligo(dT); (2) add Fragmentation Buffer to randomly interrupt the mRNA; (3) ) Using mRNA as a template, use six base random primers (random hexamers) to synthesize the first cDNA strand, then add buffer, dN TPs, RNase H and DNA polymerase I to synthesize the second cDNA strand, and use A MPure XP beads to purify cDNA; (4) The purified double-stranded cDNA is subjected to end repair, A-tailing, and sequencing adapters, and then AMPure XP beads are used for fragment size selection; (5) Finally, a cDNA library is obtained by PCR enrichment.

[0027] After the library was constructed, the library concentration and insert size (Insert Size) were detected using Qubit2.0 and Agilent 2100 respectively, and the effective concen...

Embodiment 3

[0028] Annotation of Example 3 Functional Genes

[0029] Use BLAST software to compare the Unigene sequence with NR, Swiss-Prot, GO, COG, KOG, eggNOG4.5, KEGG databases, use KOBAS2.0 to get the KEGG Orthology results of U nigene in KEGG, and predict the amino acid sequence of Unigene Then use the HMMER software to compare with the Pfam database to obtain Unigene annotation information. A total of 24,978 unigenes were annotated, of which 6,360 unigenes were annotated to the COG database, 9,961 were annotated to the GO database, 13,170 were annotated to the KEGG database, 15,484 were annotated to the KOG database, and 17,385 were annotated to the Pfam database , 13,515 were annotated to the Swiss-Prot database, 23,179 were annotated to the eggNOG database, and 23,588 were annotated to the Nr database.

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PUM

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Abstract

The invention discloses an acipenser dabryanus gonad differential expression gene expression gene GSDF and a screening method thereof, and belongs to the technical field of biology. A high-flux sequencing technique is used for performing male and female transcriptome comparative sequencing on acipenser dabryanus; transcriptome data is subjected to assembling, annotation and differential gene expression analysis. An FPKM method is used for analyzing the gene expression level in male and female gonad. EBSeq is used for performing gene differential expression analysis, so that a differential expression gene set between the male and female samples is obtained. In the screening process, FDR is selected as a key index of the differential expression gene screening; the conditions that the FDR issmaller than 0.05 and the differential multiple is greater than or equal to 2 are used as screening standards. Then, the differential gene is subjected to function annotation; the sex determination-related gene is screened; the basis is provided for the acipenser dabryanus sex determination and the sex determination gene and sex determination mechanism study in future.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a gonad differentially expressed gene GSDF of Dabry's sturgeon and a screening method thereof. Background technique [0002] The acquisition of fish sex-related information is extremely important for both the protection of its population resources and the enhancement of breeding. However, many fish have no obvious external sexual characteristics, especially in the non-breeding season, which brings great difficulties to the identification of males and females and the distinction of gonad developmental stages. Sturgeon is the oldest fish among the existing fishes. It has a history of more than 200 million years and is known as the "living fossil in water". At present, on the one hand, due to overfishing, pollution and habitat degradation, the number of wild sturgeons worldwide has declined sharply, and most of them have been listed as endangered species. On the other hand...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6869
CPCC12Q1/6869C12Q2535/122C12Q2539/113
Inventor 陈叶雨刘亚龙治海林珏龚全赖见生杜军宋明江
Owner FISHERIES INST SICHUAN ACADEMY OF AGRI SCI
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