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Refractory dihydroorate synthase gene and its polypeptide coded by it and preparing process

A technology of dihydroorotic acid and high temperature resistance, applied in the field of genetic engineering, can solve problems such as the inability to produce DHPS

Inactive Publication Date: 2002-11-13
HUADA GENE RES & DEV CENT HANGZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although, higher eukaryotic cells cannot resynthesize folic acid and thus cannot produce DHPS (dihydroorotate synthase)

Method used

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  • Refractory dihydroorate synthase gene and its polypeptide coded by it and preparing process
  • Refractory dihydroorate synthase gene and its polypeptide coded by it and preparing process
  • Refractory dihydroorate synthase gene and its polypeptide coded by it and preparing process

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Experimental program
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Effect test

Embodiment 1

[0033] 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 digestion. After the processed samples were treated with blunt...

Embodiment 2

[0034] Example 2: Genome Sequencing

[0035] 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 ABI 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

[0036] Embodiment 3: data analysis 1) Basecalling and sequencing quality monitoring

[0037] 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.

[0038] 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.

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Abstract

A process for preparing refractory dihydroorate synthase gene and the polypeptide for coding it and the said gene used for preparing its transgenic microbe or animal and plant and recovering the enzyme coded by it, and in addition, the amino acid sequence of the polypeptide with the activity of the said refractory dihydroorate synthase and its functional analog, and the process based on the wholegenom sequencing and analyzing of thermophilic anaerobe for preparing, separating and purifying the said polypeptide are disclosed. It also relates to coding of the separated DNA with the mentioned activity or its function identical variant and use of the recombinant DNA technique with the said DNA to produce the said polypeptide or its function identical variant.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a high-temperature resistant dihydroorotate synthase gene and its encoded polypeptide and a method for preparing the polypeptide. Background technique [0002] Dihydropteroate synthase (Dihydropteroate synthase) is an enzyme that plays an important role in folic acid biosynthesis. Its role is to catalyze 2-amino-4-hydroxyl 6-hydroxymethyl 7,8-dihydropteridine diphosphate and 4-aminobenzoate to generate diphosphoric acid and dihydroorotic acid. [0003] Dihydroorotate synthase is an enzyme that plays an important role in folic acid biosynthesis. Although, higher eukaryotic cells cannot de novo synthesize folic acid and therefore cannot produce DHPS (dihydroorotate synthase). DHPS acts like 4-aminobenzoate hydroxylase, which acts to create compounds and mediate pathways linked to neurodegenerative disorders. Folate deficiency has been used to study th...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/63
Inventor 董伟杨焕明卢小羽胡松年张小英
Owner HUADA GENE RES & DEV CENT HANGZHOU