Pseudobagrus-fulvidraco-richardson-origin transgenic vectors constructed with elements of pseudobagrus fulvidraco richardson [beta]-actin promoter and growth hormone gene, and application thereof

A technology of actin and yellow catfish, which is applied in the direction of using vectors to introduce foreign genetic material, recombinant DNA technology, etc.

Inactive Publication Date: 2014-04-16
NANJING UNIV
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  • Summary
  • Abstract
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  • Claims
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Problems solved by technology

However, the research and development of transgenic yellow catfish has not yet been involved in literature and research, so the construction of the original transgenic vector of yellow catfish has a broad prospect of economic benefits

Method used

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  • Pseudobagrus-fulvidraco-richardson-origin transgenic vectors constructed with elements of pseudobagrus fulvidraco richardson [beta]-actin promoter and growth hormone gene, and application thereof
  • Pseudobagrus-fulvidraco-richardson-origin transgenic vectors constructed with elements of pseudobagrus fulvidraco richardson [beta]-actin promoter and growth hormone gene, and application thereof
  • Pseudobagrus-fulvidraco-richardson-origin transgenic vectors constructed with elements of pseudobagrus fulvidraco richardson [beta]-actin promoter and growth hormone gene, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The cloning of embodiment 1 yellow catfish β-actin promoter

[0034] 1. Primer design: compare the β-actin promoter sequences of fish such as stingcatfish and tilapia published on GenBank, and design an upstream primer P1 in the homologous conserved region, which is located near the CAATbox. Afterwards, the sequences of the exons were compared, and a downstream primer P2 was designed in the conserved region of exon 3 of β-actin, and the β-actin promoter of the yellow catfish was cloned through this pair of primers .

[0035] P1: 5'-GTGWGTGACGCMGGACCAATC-3' (W=A+T, M=T+C)

[0036] P2: 5'-CCATXTCXTCCCAGTTGGTYACAAT-3' (X=A+G, Y=G+C)

[0037] 2. Genomic DNA extraction: Take the adult yellow catfish pituitary gland and cut it into 1.5ml sterilized centrifuge tube, add a certain amount (m / v=1 / 10, preferably 10ml per g tissue) of digestion buffer (10mM Tris-Cl, pH8.0, 1% SDS, 100μg / ml proteinase K) and placed in a 37°C water bath for digestion with slight shaking for 5 hour...

Embodiment 2

[0052] Example 2 Identification of the activity of the yellow catfish β-actin promoter

[0053] 1. The connection of the promoter and the yellow fluorescent protein YFP carrier: in order to prove the activity of the yellow catfish β-actin promoter, the plasmid (pActin proexon-T) constructed in Example 1 is extracted, the promoter is separated, and inserted into In the yellow fluorescent protein carrier, see if it can drive the expression of fluorescent protein. In the pActin proexon-T plasmid, the pMD18-T vector has its own BamHI site; and at the end of the inserted yellow catfish β-actin promoter, it is the beginning of encoding β-actin At the codon (ATG), it has an Nco I site. Double digestion with BamH I and Nco I can obtain a complete promoter fragment without actin sequence. In addition, the pCYP26A1-2.5keYFP plasmid was digested with BamH I and Nco I to obtain the yellow fluorescent protein vector. Enzyme digestion system: 10×buffer2.5μl, plasmid 4μl, BamH I and Nco I...

Embodiment 3

[0055] Cloning of the open reading frame (ORF) of embodiment 3 yellow catfish growth hormone gene

[0056] 1. Primer design: Design a pair of degenerate primers (P3 and P4) in the homologous conserved region with reference to the growth hormone gene cDNA sequences of fish sting catfish (AF147792), giant catfish (L27835) and channel catfish (AF267989) published in GenBank ) amplifies the open reading frame (ORF) sequence of the gene.

[0057] P3: 5'ATGGCTXGAGYYTTMGTG3' (X=A,C; Y=G,T; M=G,A);

[0058] P4: 5' CTACAGPGTGCAGTTGGAATC3' (P = G, A).

[0059] 2. Reverse transcription reaction: Take about 1 gram of brain tissue of yellow catfish and extract total RNA. For specific steps, refer to the instructions of Qiagen RNA extraction kit. The obtained total RNA was detected by 0.8% agarose gel electrophoresis and stored at -20°C for future use. The reverse transcription of RNA was carried out according to the instructions of Promega's M-MLV reverse transcriptase, and the first-st...

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Abstract

The invention discloses a [beta]-actin promoter, a growth hormone gene and a 3<,>-untranslated region (3<,> UTR) of pseudobagrus fulvidraco richardson, and a pseudobagrus-fulvidraco-richardson-origin transgenic vector constructed with the [beta]-actin promoter, the growth hormone gene, and the 3<,> UTR as elements. According to the invention, sequences of the pseudobagrus fulvidraco richardson [beta]-actin promoter, sequences of exon 1, sequences of partial exon 2, amino acid data-encoding sequences of the growth hormone gene, and sequences of the 3<,> UTR are acquired. The pseudobagrus-fulvidraco-richardson-origin transgenic vector is constructed with the obtained [beta]-actin promoter, the obtained growth hormone gene, and the obtained 3<,> UTR as elements. According to the invention, the transgenic vector is constructed through driving the growth hormone gene expression of pseudobagrus fulvidraco richardson by the [beta]-actin promoter of its own. With the technical scheme of the present invention, biosafety problems of faced by transgenic fish can be effectively avoided. Therefore, the invention has great practical significance and application prospects in the genetic breeding and artificial fertilizing of pseudobagrus fulvidraco richardson.

Description

technical field [0001] The present invention relates to the cloning, recombination and promoter activity analysis and application of the promoter of the β-actin of the yellow catfish, the growth hormone gene and the 3'-untranslated region sequence, and constructs the original transgene of the yellow catfish as an element The carrier belongs to the field of genetic engineering. Background technique [0002] Yellow catfish (Pseudobagrus fulvidraco Richardson) is commonly known as Angzi, yellow wax ding. Belongs to Siluriformes, Cullidae, Peltridae. The fish is a warm-water fish that lives on the bottom. The individual is usually about 30-100 grams, and it is widely distributed in inland waters of my country. Its body surface is smooth and phosphorus-free, the meat is tender, without intermuscular spines, the protein content is 15.37%, the total amino acid content is 14.19%, the essential amino acid index reaches 73.34, the lysine content is high, the taste is delicious, and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85
Inventor 宋伟熊熙文赵庆顺
Owner NANJING UNIV
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