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Promoter for heat shock protein gene of litopenaeus vannamei

A heat shock protein and promoter technology, applied in DNA/RNA fragments, using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problems of non-transgenic applications, achieve high biological safety, improve screening efficiency, Effects that are easy to observe and detect

Inactive Publication Date: 2012-10-31
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Currently, there is no transgenic application in Litopenaeus vannamei

Method used

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  • Promoter for heat shock protein gene of litopenaeus vannamei
  • Promoter for heat shock protein gene of litopenaeus vannamei
  • Promoter for heat shock protein gene of litopenaeus vannamei

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Screening of BAC clones containing the heat shock protein gene hsc70 of Litopenaeus vannamei

[0023] The construction of Litopenaeus vannamei BAC library clone pool comprises the following steps:

[0024] (1) Construction of plate pool: with a 384-well plate as a unit, 384 clones were mixed in equal amounts, and the obtained mixed clones were a plate pool.

[0025] (2) Construction of row pool and column pool: 24 clones in each row of the 384-well plate are mixed in equal amounts, and the obtained mixed clones form a row pool, and 16 clones in each column are mixed in equal amounts, and the obtained mixed clones form a column pool.

[0026] (3) Construction of super pools: Arrange plate pools in a certain order into 96-well plates, and then construct row pools and column pools for these plate pools arranged on 96-well plates.

[0027] Screening of positive clones containing heat shock protein gene of Litopenaeus vannamei:

[0028] The forward primer 5'-CCC...

Embodiment 2

[0082] Example 2 Cloning of the promoter fragment of the heat shock protein gene of Litopenaeus vannamei

[0083] The measured full length of the BAC clone was analyzed, and the first base of the heat shock protein gene hsc70 was recorded as +1. Take the base sequence from -3800 to 0 for promoter prediction. Three promoter regions with high scores (0.701, 0.773 and 0.696, respectively) were predicted at -700, -1600 and -3100 bases, respectively. According to the homology comparison, the gene is still transcribed at -1200 base, so the base sequence between -3700 and -1200 is determined as the promoter region of the heat shock protein gene.

[0084] Primers were designed on both sides of the promoter, and different uncut sites (Xho I and BamH I) were added, and the promoter region was amplified by polymerase chain reaction (PCR). Design forward primer F1: 5'-CTCGAGTATGCGTCAGTGCGAGTGTG-3' and reverse primer R1: 5'-GGATCCCAAATAGATCATTGGCTTACCTTAT-3' and perform polymerase chain ...

Embodiment 3

[0089] Example 3 Construction of promoter expression vector and transgenic vector pLvHscP1-EGFP

[0090] The extracted plasmid was double digested with restriction endonucleases Xho I and BamH I, and the promoter fragment in the sequence listing shown in SEQ ID NO 1. was reclaimed;

[0091] The vector pEGFP-1 was double digested with restriction enzymes Xho I and BamH I, and the linearized pEGFP-1 vector was recovered;

[0092] The recovered promoter fragment and pEGFP-1 vector were ligated overnight with T4 ligase. The connection system is as follows, and the molar ratio of pEGFP-1 to the promoter fragment is controlled between 1:2-6. The ligation product was transformed into DH5α Escherichia coli strain, spread on a plate containing kanamycin, and cultured overnight at 37°C. Select positive clones containing recombinants. Simultaneous detection using pEGFP-1 vector primers and promoter-specific primers.

[0093] Ligation system: T4 ligase 1 μl; 10x Buffer 1 μl; pEGFP-1 1...

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Abstract

The invention relates to the biotechnical field, in particular to a promoter for a heat shock protein gene of litopenaeus vannamei. The promoter for the heat shock protein gene of the litopenaeus vannamei is obtained by bacterial artificial chromosome (BAC) clone screening and sequencing analysis, promoter prediction, DNA amplification and sequencing, vector construction and other technologies, and has the base sequence at the sequence table SEQ ID NO.1; then, the cloned promoter is combined with a plasmid carrying an enhanced green fluorescence protein (EGFP) to construct an EGFP gene expression vector containing a litopenaeus vannamei autochthonous promoter; and the expression vector is transfected to an insect cell sf9 to observe that the green fluorescence protein has a strong expression and can have a higher expression after heat shock. The promoter for the heat shock protein gene of the litopenaeus vanname is obtained by a clone full sequencing method for the BAC containing a target gene and the promoter prediction method, which are more simple and fast than the conventional chromosome walking method.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the promoter of heat shock protein gene of Litopenaeus vannamei. Background technique [0002] Litopenaeus vannamei, commonly known as Penaeus vannamei, is one of the three most important economic prawns in the world, and it is also an important marine culture species in my country. It currently accounts for more than 80% of the total prawn production in my country. However, the current aquaculture industry of Litopenaeus vannamei has the problems of germplasm degradation and frequent disease occurrence, especially the large-scale outbreak of white shift syndrome virus (WSSV) of prawns, which seriously restricts the healthy development of Litopenaeus vannamei aquaculture industry in China. [0003] Transgenic is an important means of species germplasm improvement, which generally refers to the introduction of artificially constructed gene functional elements into the genome of an org...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63
Inventor 相建海赵翠郇聘张晓军李富花
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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