Malic dehydrogenase PbMDH as well as coded sequence and application thereof

A technology of malate dehydrogenase and nucleotide sequence, which is applied in the field of genetic engineering and enzyme engineering, and can solve the problems of uncommon mining of malate dehydrogenase

Active Publication Date: 2019-02-15
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the excavation of malate dehydrogenase from marine sources is rare

Method used

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  • Malic dehydrogenase PbMDH as well as coded sequence and application thereof
  • Malic dehydrogenase PbMDH as well as coded sequence and application thereof
  • Malic dehydrogenase PbMDH as well as coded sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Activation and cultivation of embodiment 1 Pseudomonas betel (Pseudomonas beteli)

[0063] Transfer Pseudomonas beteli (Pseudomonas beteli) stored at -80°C into 10 mL of 2216L medium according to 1% inoculum amount, place it in a constant temperature shaker at a temperature of 25°C and a speed of 200 rpm, and activate it for 12 hours . The activated strain was inoculated into 50 mL of 2216L medium at a ratio of 1%, and cultured in a constant temperature shaker at a temperature of 25° C. and a rotation speed of 200 rpm until the OD600 was about 0.8.

Embodiment 2

[0064] Example 2 Gene cloning of Pseudomonas beteli malate dehydrogenase PbMDH and construction of recombinant vector

[0065] The total RNA of Pseudomonas beteli was extracted using the genome extraction kit produced by Takara Company, and then cDNA was synthesized by reverse transcription, and 1 μL of cDNA was used as a template for polymerase chain reaction.

[0066] According to the nucleotide sequence of SEQ ID NO.2, a pair of primers are designed for PCR amplification, and the primers are designed as follows:

[0067] Upstream primer PbMDHF1 (SEQ ID No.9): 5'-GGAATTC CATATG TCHGATYTYAAAACYGCYG-3';

[0068] Downstream primer PbMDHR1 (SEQ ID No.10): 5'-CCG CTCGAG TYARCCSTTGAASACRTCRTC-3';

[0069] Wherein, YRS represents a degenerate base, specifically Y=C / T, R=A / G, S=G / C, and the underlined sequence is the restriction endonuclease site NdeI and XhoI;

[0070] Using the extracted Pseudomonas beteli genome cDNA as a template, the PCR amplification system is as follows...

Embodiment 3

[0081] The construction of embodiment 3 recombinant escherichia coli

[0082] The specific steps of transferring the successfully connected recombinant plasmid into Escherichia coli are as follows:

[0083] (1) Take 100 μL of competent cells and place them in an ice bath to melt for 10 minutes;

[0084] (2) Add 0.1ng-10ng (3μL-10μL) of the transformed recombinant plasmid to the competent cells, mix gently and place on ice for 30min;

[0085] (3) Place the above bacterial solution at 45°C for heat shock for 45 seconds, then quickly put it in an ice bath for 1-2 minutes;

[0086] (4) Add 890 μL fresh LB medium preheated at 37°C, and incubate at 37°C, 200 rpm for 1 hour;

[0087] (5) Spread 100-200 μL of the bacterial solution evenly on an LB plate containing kanamycin (100 μg / mL) with a coating stick, and culture overnight at 37°C;

[0088] Pick the transformants on the plate for colony PCR, screen positive clones, and the PCR results are as follows: figure 1 As shown, the s...

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Abstract

The invention provides malic dehydrogenase PbMDH derived from Pseudomonas beteli, as well as a coded gene and an application of the malic dehydrogenase. Cloned plasmid with the malic dehydrogenase PbMDH nucleotide sequence can be transferred into engineering bacteria by transduction, transformation and conjugal transfer, the malic dehydrogenase PbMDH can be efficiently expressed by adjusting expression of the coded gene, and an effective way is provided for production of the malic dehydrogenase. The marine-derived malic dehydrogenase is resistant to salt and high temperature and has importantindustrial application prospects.

Description

technical field [0001] The present invention relates to the technical fields of genetic engineering and enzyme engineering, and relates to a malate dehydrogenase PbMDH and its coding sequence and application, in particular to a malate dehydrogenase of marine-sourced Pseudomonas beteli PbMDH and its coding genes and applications. Background technique [0002] Malate dehydrogenase (EC: 1.1.1.37) widely exists in animals, plants and various organisms, and is highly conserved. It plays an important role in metabolism such as circulation and amino acid synthesis. There are different approaches to the classification of malate dehydrogenase. Classified according to its distribution in eukaryotic cells, malate dehydrogenase can be mainly divided into cytoplasmic malate dehydrogenase, mitochondrial malate dehydrogenase and chloroplast malate dehydrogenase, which exist in the cytoplasm, mitochondria and In chloroplasts, in addition, subcellular localization, malate dehydrogenase wa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53B09C1/10
CPCB09C1/10C12N9/0006C12Y101/01037
Inventor 方柏山王雅丽张永辉苏洁茹
Owner XIAMEN UNIV
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