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Ectoine synthetase gene, recombinant vector, recombinant engineering bacterium and application of recombinant engineering bacterium

A technology of recombinant engineering bacteria and tetrahydropyrimidine, applied in genetic engineering, application, plant genetic improvement and other directions, can solve the problems of ES enzyme gene cloning difficulty, low similarity, etc., and achieve extensive social and environmental benefits, good treatment effect , good stability

Active Publication Date: 2012-06-27
SHANDONG WEIFANG RAINBOW CHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Due to the low similarity of the amino acid sequence of the ES enzyme gene, that is, there are large differences in the ES enzyme gene sequence between microorganisms of different species or even different genera of the same species, so the cloning of the ES enzyme gene is relatively difficult.

Method used

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  • Ectoine synthetase gene, recombinant vector, recombinant engineering bacterium and application of recombinant engineering bacterium
  • Ectoine synthetase gene, recombinant vector, recombinant engineering bacterium and application of recombinant engineering bacterium
  • Ectoine synthetase gene, recombinant vector, recombinant engineering bacterium and application of recombinant engineering bacterium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The original process of obtaining the ES enzyme gene of the present invention is as follows:

[0054] The One-4-All Genomic DNA Mini-Preps Kit of Shanghai Sangon was used to extract the genomic DNA of Bacillus (Bacillaceae) (see the kit manual for the operation steps). According to the amino acid sequence of ectoine synthase, degenerate primer sequences were designed, the upstream primer was GGNTTYWSNTTYCAYATHACN, and the downstream primer was NGGRTTRAANACRCA. Then, using the bacillus genomic DNA as a template, the ES enzyme gene containing restriction endonuclease (EcoRI and NotI) enzyme cutting sites is obtained by PCR under the effect of degenerate primers, and its nucleotide sequence is as SEQ: NO .1 shown.

[0055] reaction system:

[0056] 10× buffer 5.0 μL

[0057] dNTPs (2.5 mM) 4.0 μL

[0058] Upstream primer (50 mM) 1.0 μL

[0059] Downstream primer (50 mM) 1.0 μL

[0060] Taq DNA polymerase 0.5 μL

[0061] Bacillus genomic DNA 1.0 μL

[0062] h 2 O 3...

Embodiment 2

[0066] On the basis of obtaining the nucleotide sequence of the ES enzyme gene of the present invention, the acquisition of its gene can be obtained by cloning by existing methods, and its method is as follows:

[0067] 1. Cloning of ES enzyme gene for expression

[0068] According to the SEQ NO:1 gene sequence obtained in Example 1, design and synthesize amplification primers Qe-1 and Qe-2.

[0069] Qe-1 5'-GGCCGTTCTGGCCGCAGGCGTGTCTCAAGG-3'

[0070] Qe-2 5'-GGATCCGTTCTCAACTTGTG-3'

[0071] Using Bacillus genomic DNA as a template, PCR amplification is carried out under the action of primers, and the reaction system is:

[0072] 10× buffer 5.0 μL

[0073] dNTPs (2.5 mM) 4.0 μL

[0074] Qe-1 (50 mM) 1.0 μL

[0075] Qe-2 (50 mM) 1.0 μL

[0076] Taq DNA polymerase 0.5 μL

[0077] Bacillus genomic DNA 1.0 μL

[0078] Double distilled water 37.5 μL

[0079] Using PCR instrument, reaction conditions: pre-denaturation at 94 oC for 5 min; denaturation temperature at 94 oC for...

Embodiment 3

[0082] The gene fragment of the present invention can be connected with a plasmid to construct a recombinant expression vector, and the method is as follows:

[0083] Recover the gene fragment of the above-mentioned Example 2 and link it with the T vector pBST (Tiangen Company), transform it into Escherichia coli (Escherichiacoli) DH5α, screen for resistance, extract a plasmid that meets the requirements, and record it as pBST-ES, and the resulting plasmid and expression vector pIC9K (its plasmid map is shown in Figure 4 shown) to carry out double enzyme digestion respectively, the double enzyme digestion reaction system is as follows:

[0084] pIC9K or pBST-ES 10 μL

[0085] 10×H buffer 2 μL

[0086] EcoRI endonuclease 1 μL

[0087] NotI endonuclease 1 μL

[0088] Double distilled water 6 μL

[0089] Reaction conditions: digest at 37oC for 2-4 hours, add 4 μL of 10×loading buffer to terminate the reaction, detect the reaction solution by 1% agarose gel electrophoresis, ...

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Abstract

The invention discloses an ectoine synthetase gene, a recombinant vector, a recombinant engineering bacterium and an application of the engineering bacterium. The gene which can code ectoine synthetases is efficiently expressed in host cells to produce the ectoine synthetases, so the engineering bacterium containing the gene, which has the advantages of stable structure, excellent performances, high salt tolerance, no need of domestication during high salt wastewater processing, high salt wastewater processing cost reduction, and production technology simplification, can be widely applied to the high salt wastewater processing in the daily chemical industry, the food processing industry, the leather processing industry, the organic synthetic industry, the medicine preparation industry andthe like, and has great meanings.

Description

technical field [0001] The invention relates to an ectoine synthase gene, a recombinant carrier, a recombinant engineering bacterium and an application thereof, and belongs to the field of genetic engineering of enzymes. Background technique [0002] Tetrahydropyrimidine as a compatible solute in extreme halophilic bacteria in 1985 Ectothiorhodospira halochloris It was discovered and identified by Galinski et al. In 1988, Inbar et al. in Gram-positive soil bacteria Streptomyces parvulus Hydroxylated derivatives of ectoine were found in Hydroxylated ectoine. Ecs is one of the most common compatible solutes in halophilic bacteria, which can provide cells, proteins, Cell membranes and nucleic acids provide protection, so they are widely concerned by researchers from all over the world. In addition, Ecs has a certain effect on neurological diseases such as Alzheimer's disease and Parkinson's disease, and the latest research has found that Ecs can improve skin regeneration an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/63C12N15/74C12N1/21C02F9/14C12R1/01C12R1/07C02F101/34C02F101/36
Inventor 孙国庆李志清陈蕾凌晓光
Owner SHANDONG WEIFANG RAINBOW CHEM
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