Goldfish autonomous transposon gene gfTol2, transposase coded by gene, and use of gene

A technology of transposase and transposon, applied in gene therapy, enzyme, micro-injection method, etc., can solve the problems of insufficient quantity of finished products, affecting the reputation of Chinese goldfish, destroying the brand of Chinese goldfish, etc., reducing manpower and improving benefit, increase the effect of insertional mutagenesis

Inactive Publication Date: 2010-04-28
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This not only wastes a lot of manpower and material resources, but also the quantity of finished products cannot meet the demand of the international market
In order to make up enough quanti

Method used

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  • Goldfish autonomous transposon gene gfTol2, transposase coded by gene, and use of gene
  • Goldfish autonomous transposon gene gfTol2, transposase coded by gene, and use of gene
  • Goldfish autonomous transposon gene gfTol2, transposase coded by gene, and use of gene

Examples

Experimental program
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Embodiment 1

[0037] Example 1. Isolation of goldfish autonomous transposon gene gfTol2

[0038] By comparing the amino acid sequences of maize AC transposon (GenBank accession number: P08770) and medaka Tol2 transposon (GenBank accession number: AB031079.1), we found that the second coding region of maize AC and medaka Tol2 transposon On the second coding region of the transposon, there are two amino acid sequences with a similarity of 100%, wherein the size of the first amino acid similar fragment is 9 amino acids (CACHILNLV), and the second amino acid similar fragment is 6 amino acids ( TRWNST) sequence. According to the nucleotide sequences of these two amino acid conservative sequences, primers SEQ ID No: 3 and SEQ ID No: 4 were designed respectively, and this pair of primers (SEQ ID No: 3 and SEQ ID No: 4) was used for hAT family transposition For the amplification of sub-small fragments, the PCR reaction program is: 94°C for 5min; 94°C for 30s, 55°C for 30s, 72°C for 60s, 35 cycles;...

Embodiment 2

[0043] Embodiment 2, the cloning of full-length cDNA of goldfish gfTol2 transposase

[0044] For Ryukin goldfish derived from embryos of different periods, 10 samples were taken to extract total RNA with TRIZOL (Invitrogen) reagent, reverse transcriptase in TaKaRa RNA PCR Kit (AMV) Ver. A pair of forward and reverse primers (SEQ ID No: 12 and SEQ ID No: 13) amplified a small fragment of goldfish gfTol2, and then cloned and sequenced it (commissioned by Beijing BGI Gene Research Center) to determine whether there is expression of gfTol2 gene. 3'- and 5'-RACE amplification of embryonic phase cDNA with gfTol2 gene expression.

[0045] 3'-RACE: Design 3'-RACE primers (SEQ ID No: 14) and 3'-RACE nested primers (SEQ ID No: 15) according to the measured small fragment sequence, using the 3'-end single-stranded cDNA as a template, Use the M13Primer M4 (SEQ ID No: 16) in the 3'-RACE kit as a reverse primer, and the 3'-RACE primer (SEQ ID No: 14) as a forward primer for PCR amplificati...

Embodiment 3

[0048] Embodiment 3, the relationship between gfTol2 gene overexpression and goldfish variation

[0049] For Ryukin goldfish derived from embryos of different periods, 10 samples were taken to extract total RNA with TRIZOL (Invitrogen) reagent, reverse transcriptase in TaKaRa RNA PCR Kit (AMV) Ver. 1 pair of forward and reverse primers (SEQ ID No: 19 and SEQ ID No: 20) amplifies the full-length cDNA of goldfish gfTol2, the PCR reaction program is: 94°C for 5min; 94°C for 30s, 55°C for 30s, 72°C for 60s, 35 Cycle; 72°C extension for 5 min. After the PCR product was digested with BamHI and XbaI, it was subcloned into the pCS2+ vector which was digested with BamHI and XbaI. After the pCS2+ recombinant plasmid was linearized by NotI, gfTol2 mRNA was synthesized using the Sp6 mMessage mMachine kit (Ambion) kit. The obtained gfTol2mRNA was injected into 1-2-cell stage Ryukin goldfish embryos by microinjection, each embryo was injected with 1nl gfTol2mRNA solution, and the injectio...

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Abstract

The invention discloses a goldfish autonomous transposon gene gfTol2, which has a nucleotide sequence shown as SEQ ID No:1. The invention also discloses a transposase coded by the gene gfTol2, which has an amino acid sequence shown as SEQ ID No:2. The invention also discloses use of the gene gfTol2 as a gene medicament or a gene reagent for goldfish; and the rate of graded product of the goldfish is improved and offspring mutation is induced by adjusting the concentration of a transcription product of the gene.

Description

technical field [0001] The invention relates to a goldfish autonomous transposon gene gfIol2, the transposase encoded by the gene and the application of the gene. The invention relates to the separation of the goldfish autonomous transposon gene and the separation of the cDNA encoded by the transposase. The gene product is used to regulate the authenticity rate of goldfish and induce variation in offspring. The invention belongs to the field of fish genetic engineering. Background technique [0002] Transposons are mainly divided into two categories: one is type I transposons with a structure similar to retroviruses, using the "copy and paste" mechanism, first transcribing DNA into RNA, and synthesizing DNA under the action of reverse transcriptase , The reverse-transcribed DNA is inserted into other parts of the chromosome, and the position on the chromosome is shifted as a result. The other type is type II transposon, which mainly adopts the "cut-paste" transposition mec...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N9/00C12N15/89A61K48/00
Inventor 邹曙明蒋霞云何为袁剑杜雪地沈睿杰
Owner SHANGHAI OCEAN UNIV
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