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Cordyceps sinensis ribonucleotide reductase as well as coding gene and application thereof

A Cordyceps sinensis ribonucleotide and reductase technology, applied in the directions of oxidoreductase, application, genetic engineering, etc., can solve the problems of rare gene and protein research, and achieve major application prospects, high expressivity, and the effect of expanding production.

Inactive Publication Date: 2013-04-17
ZHEJIANG UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Cordyceps sinensis, as an important synthetic metabolite of pyrimidine nucleosides, still only stays in the research of metabolite composition analysis and efficacy, and the research on the related genes and proteins in the synthetic metabolic pathway of pyrimidine nucleosides of Cordyceps sinensis is still rare

Method used

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  • Cordyceps sinensis ribonucleotide reductase as well as coding gene and application thereof
  • Cordyceps sinensis ribonucleotide reductase as well as coding gene and application thereof
  • Cordyceps sinensis ribonucleotide reductase as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Cultivation of "Bailing" Production Fungus Cordyceps sinensis Trichosporium

[0032] Strain source: First, collect natural Cordyceps sinensis from Qinghai, and bring it back to Hangzhou for isolation and screening. The L0106 strain was obtained, and the strain was identified as Hirsutella Sinensis, which is preserved in China's typical culture. The depository number is CCTCC No: M 2011278, which has been disclosed in the previously applied patent CN102373190A.

[0033] The strain is inoculated on the slant, and the medium formula (this is the liquid formula before solidification, and the slant will be made after being prepared according to the following ratio) is glucose 2.0% (w / v, 1% means that 100mL medium contains 1g , the same below), corn flour 1.0%, potato juice 0.5%, dextrin 0.5%, yeast powder 0.5%, bran 1.0%, silkworm chrysalis powder 2.0%, peptone 1.0%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.05% , agar powder 1.0%, the balance is ...

Embodiment 2

[0034] Example 2: Extraction of total RNA from Trichospora chinensis from "Bailing" producing bacteria Cordyceps sinensis

[0035] Extract total RNA with TRIzol reagent. The steps are as follows: 1) Grinding in liquid nitrogen: Take 1 g of fresh bacterial cells into a mortar, add liquid nitrogen repeatedly to fully grind to a powdery state, and distribute them into pre-cooled 1.5 mL centrifuge tubes. Add 1 mL of TRIzol reagent, mix well, and let stand on ice for 5 min to completely separate the nucleic acid-protein complex. 2) RNA isolation: add 0.2 mL of chloroform, shake vigorously and mix for 15 s, stand on ice for 2-3 min, centrifuge at 4°C and 12000 rpm for 15 min, separate layers, and take the upper aqueous phase, about 600 μL. 3) RNA precipitation: add 500 μL of isopropanol, stand on ice for 10 min, centrifuge at 4°C and 12000 rpm for 10 min, and discard the supernatant. 4) RNA washing: add 1 mL of 75% (v / v) ethanol, suspend the precipitate, stand on ice for 10 min, an...

Embodiment 3

[0036] Example 3: Sequencing of RNA samples of "Bailing" producing fungus Cordyceps sinensis

[0037] After extraction of total RNA from the sample, mRNA was enriched with magnetic beads with Oligo(dT). Add fragmentation buffer to break mRNA into short fragments (200~700bp), use mRNA as template, use 6-base random primers (random hexamers) to synthesize the first cDNA chain, then synthesize the second cDNA chain, and then go through QiaQuick PCR The kit was purified and eluted with EB buffer for end repair, polyA was added, and sequencing adapters were connected, then agarose gel electrophoresis was used for fragment size selection, and finally PCR amplification was performed. The constructed sequencing library was performed with Illumina GA IIx Sequencing. The raw image data obtained by sequencing is converted into sequence data by base calling, that is, raw data or raw reads. The reads containing only adapter sequences in the original sequencing reads were removed for subs...

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Abstract

The invention relates to ribonucleotide reductase from 'Bailing' producing strain cordyceps sinensis hirsutella sinensis for anabolism of deoxidized pyrimidine nucleoside diphosphate from pyrimidine nucleoside diphosphate, a gene coding the ribonucleotide reductase and application of the ribonucleotide reductase. The ribonucleotide reductase has over 90% of homology to the amino acid sequence shown by SEQ ID No.1 or SEQ ID No.3; and the coding gene has over 90% of homology to the nucleotide sequence shown by SEQ ID No.2 or SEQ ID No.4. According to the invention, the metabolic pathway for synthesizing deoxidized pyrimidine nucleoside diphosphate from pyrimidine nucleoside diphosphate is studied in details in principle, the cloning DNA (Deoxyribonucleic Acid) comprising the nucleotide sequence provided by the invention can be transferred into engineering bacteria through transduction, transformation and combined transfer, high expressivity of the host deoxidized pyrimidine nucleoside diphosphate is obtained by adjusting the expression of the deoxidized pyrimidine nucleoside diphosphate biosynthesis gene, an effective means of increasing output of the deoxidized pyrimidine nucleoside diphosphate is provided, and a significant application prospect is realized.

Description

(1) Technical field [0001] The present invention relates to a kind of ribonucleotide reductase (ribonucleotide reductase, class II) which is involved in the synthesis and metabolism of pyrimidine diphosphate nucleoside from the production bacterium Cordyceps sinensis of Trichosporium chinensis from "Bailing" producing bacterium, and encodes this Enzyme genes and their applications. (2) Background technology [0002] Cordyceps sinensis (Berk.) Sacc. is a complex of Cordyceps sinensis fungus parasitic on the larvae of Lepidoptera (Lepidoptera) Hepialus armoricanus Oberthur and the corpse of the larva ). Cordyceps sinensis is a kind of cherished traditional fungal medicinal resources, with the characteristics of diverse metabolites and biological activities, showing great application and development prospects in the field of biomedicine. Cordyceps sinensis has attracted much attention for its wide and obvious medicinal effects, and is highly respected around the world. Chine...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12P19/30C12N15/53C12N15/70C12N1/21C12R1/645
Inventor 郑裕国柳志强吴晖李邦良许静吴玲芳许峰薛亚平袁水金王鸿艳
Owner ZHEJIANG UNIV OF TECH
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