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High-temp. resistant isocitric dehydrogenase gene, its coded polypeptide and its preparation method

A technology of isocitrate dehydrogenase and high temperature resistance, applied in the field of mutation or genetic engineering, can solve the problems of no tricarboxylic acid cycle, lack of respiratory chain, and the inability of isocitrate dehydrogenase to generate acetate, etc.

Inactive Publication Date: 2004-09-15
HUADA GENE RES & DEV CENT HANGZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in bacteria, NAD-dependent isocitrate dehydrogenase cannot generate acetate, lacking both the respiratory chain and the complete tricarboxylic acid cycle

Method used

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  • High-temp. resistant isocitric dehydrogenase gene, its coded polypeptide and its preparation method
  • High-temp. resistant isocitric dehydrogenase gene, its coded polypeptide and its preparation method
  • High-temp. resistant isocitric dehydrogenase gene, its coded polypeptide and its preparation method

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1: Construction of a sequencing library

[0035] The sequencing library was constructed using the genome-wide shotgun method. Firstly, Tengchong thermophilic anaerobic bacteria were cultured according to (Yanfen Xue, 2000) modified MB medium (Balch et al., 1979), bacteria were collected according to Marmur (1961) method, and total DNA was extracted. In order to ensure the randomness of sequencing library construction and avoid the problem of hot spots of breakage to the greatest extent, a variety of methods and principles of library construction under different conditions were adopted. Firstly, physical shearing method (including ultrasonic method and shearing with Hydroshear Machine) was used, and then AluI was selected for random partial enzyme digestion according to the genome characteristics of the bacteria. Different intensities were used to process samples during physical shearing, and samples were processed by setting enzyme gradients during enzyme diges...

Embodiment 2

[0036] Example 2: Genome Sequencing

[0037] When sequencing the genome of thermophilic anaerobic bacteria in Tengchong, two automatic sequencers were mainly used: ABI377 and MegaBACE 1000. Both sequencers use the principle of electrophoresis for sequencing (see figure 2 ), 96 samples can be completed each time. ABI377 is a product of PE Company, which is a kind of ABI series. It belongs to the slab gel electrophoresis sequencer. MegaBACE 1000 is a product of Pharmacia, which belongs to capillary gel electrophoresis sequencer.

Embodiment 3

[0038] Example 3: Basecalling and sequencing quality monitoring

[0039] The so-called basecalling refers to the process of obtaining the correct base sequence from the raw data file obtained on the sequencer. Since the sequencer obtains the intensity change traces (traces) of light of different wavelengths corresponding to the four bases A, T, G, and C, it is necessary to use a computer to adopt a certain algorithm to correctly identify the bases corresponding to the different traces. . We used Phred software (Ewing B, Hillier L, 1998) because its results are more reliable and its output is more convenient for further analysis by other programs in the same software package.

[0040] The algorithm principle of Phred's basecalling is to judge the base type based on the shape, distance, and signal-to-noise ratio of each peak in the trajectory, and at the same time give the credibility information for this base, that is, the sequencing quality of the base. In large-scale sequen...

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Abstract

The present invention discloses a high-temp. resistant isocitrate dehydrogenase gene, its coded polypeptide and its preparation method. It relates to coding separated DNA with activity and its functional identification variant and utilizing the recombinant DNa technique and using the described separated DNA to produce polypeptide with high-temp. resistant isocitrate dehydrogenase activity or its functional identification variant. This invention uses tengchaong thermophilic anaerobe hologenome sequencing and analysis as basis to clone and separate a kind of high-temp. resistant isocitrate dehydrogenase gene, and the said gene can be used for producing a transgenic microbe or animals or plant of high-temp. resistant isocitrate dehydrogenase, and recovering the enzyme coded by the said gene. Besides, this invention also provides amino acid sequence of polypeptide with high-temp. resistant another isocitrate dehydrogenase activitity and its functional identification body, and also provides the preparation, separatino and purification processes of the said polypeptide.

Description

technical field [0001] The invention relates to mutation or genetic engineering, in particular to a high-temperature-resistant isocitrate dehydrogenase gene, its coded polypeptide and its preparation method. Background technique [0002] The reaction catalyzed by isocitrate dehydrogenase is as follows: [0003] <chemistry num="001"> <chem file="01145589_cml001.xml" / > < / chemistry> [0004] <chemistry num="002"> <chem file="01145589_cml002.xml" / > < / chemistry> [0005] In the tricarboxylic acid cycle, isocitrate dehydrogenase catalyzes the reaction to produce 90% of NACPH available for biosynthesis, and then generates a large amount of acetate. In bacteria, however, NAD-dependent isocitrate dehydrogenase cannot generate acetate, lacking both the respiratory chain and the complete tricarboxylic acid cycle. The isocitrate dehydrogenase of E. coli lacks the Rossmann fold that other dehydrogenases have. [00...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/02C12N15/53C12N15/63C12Q1/68
Inventor 包其郁杨焕明林霞胡松年李蔚
Owner HUADA GENE RES & DEV CENT HANGZHOU
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