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Oreochromis niloticus alexin C9 gene clone and application thereof

A Nile tilapia, genetic technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2016-08-03
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report on complement C9 in Nile tilapia

Method used

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  • Oreochromis niloticus alexin C9 gene clone and application thereof
  • Oreochromis niloticus alexin C9 gene clone and application thereof
  • Oreochromis niloticus alexin C9 gene clone and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Obtaining the full-length cDNA of OnC9 gene

[0030] 1. Artificial insemination of Nile tilapia to obtain embryos:

[0031] Feed the male and female Nile tilapia separately in separate tanks. Observe the cloaca of the female tilapia. If the color turns red and the eggs flow out automatically when the abdomen is gently squeezed, it means that the egg cells are mature and can be artificially collected. When picking eggs, squeeze the eggs into a clean large beaker, squeeze in the semen in the same way, and stir gently with a feather to fully mix the sperm and eggs. Then add clear water, stir gently and let stand for 1 min, pour off the sewage, clean and obtain artificially inseminated tilapia roe. Fertilized eggs were cultured in an artificial incubator at 27°C.

[0032] 2. Preparation of cDNA

[0033] According to the operating instructions of the total RNA extraction reagent TrizolReagent kit (full type gold), extract the total RNA of the Nile tilapia embry...

Embodiment 2

[0037] Example 2: Sequence analysis of OnC9 gene

[0038] Use the BLAST (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) software on the NCBI website to perform a homology search on the sequence obtained after sequencing, and use the DNAMAN software to perform sequence splicing to obtain the full-length cDNA sequence of OnC9 (Sequence Listing SEQ ID NO. 1). Open reading frames were determined online using ORFFinder (http: / / www.ncbi.nlm.nih.gov / projects / gorf / ). The results showed that the full length of the OnC9 sequence was 2502bp, of which the open reading frame was 1761bp long and could encode 586 amino acids. The 5' untranslated region (Untranslated region, UTR) is 133bp long, and the 3' untranslated region is 608bp long. An mRNA instability signal ATTTA and a polyadenylation tailing signal AATAAA appeared in the 3'UTR region.

[0039] TMHMM software (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ) predicts transmembrane regions of proteins; SignalP4.1Server (http: / / www.cbs.dtu.dk / s...

Embodiment 3

[0042] Example 3: Analysis of tissue expression of OnC9 in healthy Nile tilapia

[0043]According to the cloned OnC9 cDNA sequence, primers C9-qr and C9qf for real-time fluorescent quantitative PCR were designed, and ten healthy tissues (brain, kidney, spleen, heart, liver, gills, muscle, head, etc.) Kidney, skin, thymus, intestinal tract, blood) total RNA extraction reagent TrizolReagent kit (full type gold) used to obtain total RNA, according to TransScriptAll-in-OneFirst-StandcDNASynthesisSuperMixforqPCR (One-StepgDNARemoval) reverse transcription kit (full (Formula Gold) instructions to operate reverse transcription to synthesize cDNA for quantitative PCR detection. At the same time, EF-1α was used as an internal reference gene, and EF-1αF and EF-1αR were used as primers for PCR amplification. The reaction system was: 10ul2× TopGreenqPCRSuperMix (full gold), 0.4ul of each primer, 0.4ul of passiveReferenceDye (50×), 0.4ul of ddH207.8ul, and 1.0ul of template. The reactio...

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Abstract

Oreochromis niloticus alexin C9 gene clone and application thereof. The invention belongs to functional study of the Oreochromis niloticus infected Streptococcus agalactiae. The invention discloses immune-related gene OnC9 of Oreochromis niloticus, and the nucleotide sequence is shown in the table SEQ ID NO.1. And the invention discloses the sequence of the immune-related protein OnC9 of Oreochromis niloticus, and the amino acid sequence thereof is shown in the sequence table SEQ ID NO.2. The method uses the disclosed nucleotide sequence of Oreochromis niloticus OnC9 to design a primer of fluorogenic quantitative PCR detection (C9-qf:5'-GGCTTGAAGTCGCTCTCCA-3'; C9-qr:5'-GCCAAATGCTTCTACACTTCG-3'), and carries out the fluorogenic quantitative PCR detection for the healthy Oreochromis niloticus or Oreochromis niloticus infected Streptococcus agalactiae. The method can be used for researching immune response of Oreochromis niloticus after infecting Streptococcus agalactiae.

Description

Technical field: [0001] The present invention relates to Nile tilapia (Oreochromis niloticus) gene, specifically the cloning and tissue expression analysis of immune function-related gene complement C9 (OnC9), while using real-time fluorescent quantitative PCR to identify the gene in Nile tilapia infection Function after Streptococcus agalactiae. Background technique: [0002] Tilapia, commonly known as "African crucian carp", is native to Africa, belongs to Perciformes (Perciformes), Cichlidae (Cichlidae), and is a euryhalal tropical fish. In 1956, tilapia was introduced into my country for the first time. After several introductions and genetic improvements by different units, it has become the main farmed fish in the world, and it is also the largest freshwater cultured species for export in my country. The output and export volume respectively account for About 40% and 70% of the global total. However, in recent years, the outbreak of tilapia streptococcal disease has s...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/46C12Q1/68C12N15/11
CPCC07K14/461C12Q1/6883C12Q2600/158
Inventor 曹建萌卢迈新胡欣欣陈琼刘志刚陈昆平高风英
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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