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Litopenaeus vannamei metallothionein gene LvMT as well as coding gene and application thereof

A technology of metallothionein and vannamene, applied in the field of genetic engineering, can solve the problems of MT gene identification and functional analysis that have not been reported, and achieve the effect of improving the ability to tolerate heavy metals, large economic value, and wide application prospects

Active Publication Date: 2013-04-24
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the screening of MT genes in Litopenaeus vannamei, and the identification and functional analysis of MT genes have not been reported so far.

Method used

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  • Litopenaeus vannamei metallothionein gene LvMT as well as coding gene and application thereof
  • Litopenaeus vannamei metallothionein gene LvMT as well as coding gene and application thereof
  • Litopenaeus vannamei metallothionein gene LvMT as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Cloning and sequencing of the metallothionein gene LvMT of Litopenaeus vannamei

[0028] 1. Design of primers

[0029] The full-length EST of the metallothionein gene of Litopenaeus vannamei was screened from the freshwater stress library of Litopenaeus vannamei, and the upstream primer (5'-ACG GGATCC GCCACCATGCCTGATCCATGCTGT-3') (the underline indicates the restriction site BamHI) and the downstream primer (5'-AGC AAGCTT CTGGGCAGCACTT-3') (the underline indicates the enzyme cutting site HindIII), and the primers were synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd.

[0030] 2. Extraction of RNA

[0031] Using the gill tissue of Litopenaeus vannamei as the material, the total RNA was extracted by the Trizol method, and the specific operation was as follows:

[0032] Grind 0.1 g of Litopenaeus vannamei gill tissue into powder with liquid nitrogen, add 1 mL of Trizol extract (purchased from Invitrogen, product number: 15596-026), and place it at r...

Embodiment 2

[0043] Example 2: Construction of prokaryotic recombinant expression vector pET-32a-LvMT

[0044] The recombinant vector pMD18-LvMT and the prokaryotic expression vector pET-32a (purchased from Tiangen Biochemical Technology Co., Ltd., catalog number: v1078) were double digested with BamHI restriction endonuclease and HindIII restriction endonuclease respectively to obtain the LvMT gene fragment and linear pET-32a double-digested fragments, the specific steps are: add recombinant vector pMD18-LvMT 2μl (100ng / μl), BamHI restriction enzyme 0.5μl, HindIII restriction endonuclease to 0.5mL centrifuge tube 0.5μl, 10×K buffer 2μl, using ddH 2 Make up O to 20 μl and react at 37°C for 8h. Use 1.2% agarose gel electrophoresis for detection, use an agarose gel recovery kit to recover LvMT gene and prokaryotic expression vector pET-32a, and connect the recovered fragments. The specific steps are: 3 μl (about 300ng) of LvMT gene fragments, prokaryotic Expression vector pET-32a1μl (100ng...

Embodiment 3

[0048] Example 3: Induced expression of prokaryotic recombinant expression vector pET-32a-LvMT

[0049] Take 500 μl of the Escherichia coli BL21 bacterial liquid transformed with the prokaryotic recombinant expression vector pET-32a-LvMT and the Escherichia coli BL21 bacterial liquid transformed with the empty prokaryotic expression vector pET-32a, respectively, and place them in 30 mL of LB liquid medium, shake at 37°C and 180rpm Bed cultured for 2 hours, after the culture, take out 1ml respectively, measure the OD600 value of the bacterial solution, and when the OD600 reaches 0.6, take out 2 tubes (2ml / tube) of the bacterial solution respectively, as the sample without IPTG induction and the negative control without IPTG induction , and then add IPTG with a final concentration of 0.8mM to the remaining two bacterial solutions for induction, culture on a shaker at 30°C and 180rpm for 2.5h, and take out 2 tubes (2ml / tube) of bacterial solutions as samples induced by IPTG and a...

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Abstract

The invention discloses a litopenaeus vannamei metallothionein gene LvMT as well as a coding gene and an application of the litopenaeus vannamei metallothionein gene LvMT. The nucleotide sequence of the litopenaeus vannamei metallothionein gene LvMT is shown as SEQ ID NO.1, and the amino acid sequence of the coded metallothionein gene LvMT is shown as SEQ ID NO.2. The metallothionein gene LvMT is cloned from litopenaeus vannamei for the first time, and the gene has the function of resisting heavy metals through an experimental proof, so that the litopenaeus vannamei metallothionein gene LvMT provides an effective technological means for improving the ability of tolerating heavy metals of host cells and overcoming the defect of low heavy metal tolerance of the host cells and has wide application prospect and great economic value.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the metallothionein gene LvMT of Litopenaeus vannamei and its coded protein and application. Background technique [0002] The chemical name of metallothionein (metallothionein, MT) is metallothiohistidine trimethyl inner salt, which is a kind of low molecular weight (6-7kDa), rich in cysteine, thermostable , Inducible non-enzyme protein. In 1957, Margoshes and Vallee first discovered MT in horse kidney cells; in 1977, Casterline and Barnett discovered the first MT in plants in soybean roots, and then successively discovered plants in bran grass, tomato and other plants Metallothionein, MT genes and proteins have been found in a variety of eukaryotic and prokaryotic organisms. Metallothionein is rich in Cys, can bind a large amount of divalent heavy metal ions, has strong metal binding ability and redox ability, and is mainly involved in the storage, tra...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/825C12N15/63C12N15/70C12N1/21C12R1/19
Inventor 王艳红任春华胡超群张吕平夏建军
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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