Cordyceps Chinese Hirsutella nucleoside diphosphokinase, coding gene and application thereof

A technology of nucleoside diphosphate kinase and Cordyceps sinensis of Trichoderma Effect

Active Publication Date: 2013-04-10
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

Method used

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  • Cordyceps Chinese Hirsutella nucleoside diphosphokinase, coding gene and application thereof
  • Cordyceps Chinese Hirsutella nucleoside diphosphokinase, coding gene and application thereof
  • Cordyceps Chinese Hirsutella nucleoside diphosphokinase, coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Cultivation of "Bailing" producing fungus Cordyceps sinensis

[0035] Strain source: Firstly, natural Cordyceps sinensis was collected from Qinghai and brought back to Hangzhou for isolation and screening. The strain L0106 was obtained, and the strain was identified as Hirsutella Sinensis. The strain was preserved in China for typical culture The collection number is CCTCC No: M 2011278, which has been disclosed in the previously applied patent CN102373190A.

[0036] Inoculate the strain on the slant, the medium formula (this is the liquid formula before solidification, and then make the slant according to the following ratio) is glucose 2.0% (w / v, 1% means 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 pupa powder 2.0%, peptone 1.0%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.05% , Agar powder 1.0%, the remainder is water, cultured at 12~16℃ for 25 days; then the...

Embodiment 2

[0037] Example 2: Extraction of total RNA from "Bailing" producing strain Cordyceps sinensis

[0038] Extract total RNA with TRIzol reagent, the specific steps are as follows: 1) Liquid nitrogen grinding: Take 1g of fresh bacteria into a mortar, add liquid nitrogen repeatedly to fully grind it to powder, and distribute it into pre-cooled 1.5mL centrifuge tubes. Add 1 mL of TRIzol reagent, mix well, and let stand on ice for 5 minutes to completely separate the nucleic acid protein complex. 2) RNA isolation: add 0.2mL chloroform, vigorously shake and mix for 15s, let stand on ice for 2~3min, centrifuge at 4℃, 12000rpm for 15min, layer into layers, take the upper water phase, about 600μL. 3) RNA precipitation: add 500μL of isopropanol, let stand on ice for 10min, centrifuge at 4℃, 12000rpm for 10min, discard the supernatant. 4) RNA washing: add 1mL of 75% (v / v) ethanol, suspend the precipitate, let stand on ice for 10 minutes, centrifuge at 4°C, 7500 rpm for 15 minutes; repeat the ...

Embodiment 3

[0039] Example 3: Sequencing of RNA samples of "Bailing" producing strain Cordyceps sinensis

[0040] After extracting the total RNA of the sample, the mRNA is enriched with magnetic beads with Oligo (dT). Add fragmentation buffer to break mRNA into short fragments (200~700bp), use mRNA as template, synthesize the first cDNA strand with random hexamers, then synthesize the second cDNA strand, and then go through QiaQuick PCR After the kit is purified and eluted with EB buffer, the ends are repaired, polyA is added and the sequencing adapters are added, then fragment size selection is performed by agarose gel electrophoresis, and finally PCR amplification is performed. The established sequencing library is performed with Illumina GA IIx Sequencing. The original image data obtained by sequencing is converted into sequence data by base calling, namely raw data or raw reads. Remove the reads containing only the adaptor sequence from the original sequencing reads and prepare them fo...

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Abstract

The invention relates to an enzyme from Bailing production bacterium Cordyceps Chinese Hirsutella for synthesizing metabolic (desoxy) pyrimidine nucleoside triphosphate from (desoxy) pyrimidine nucleoside diphosphate, a gene for coding the enzyme and application thereof. The enzyme is nucleoside diphosphokinase which has more than 90% of homology with amino acid sequence disclosed as SEQ ID NO.1; and the coding gene has more than 90% of homology with nucleotide sequence disclosed as SEQ ID NO.2. The invention researches the metabolic pathway of (desoxy) pyrimidine nucleoside diphosphate synthesized (desoxy) pyrimidine nucleoside triphosphate in details in principle. The cloned DNA (deoxyribonucleic acid) comprising the nucleotide sequence provided by the invention can be transformed into engineering bacterium by transduction, conversion and conjugal transfer. By adjusting the expression of the (desoxy) pyrimidine nucleoside triphosphate biosynthesized gene, the host (desoxy) pyrimidine nucleoside triphosphate is endowed with high expressivity, thereby providing an effective way for enhancing the yield of the (desoxy) pyrimidine nucleoside triphosphate and having great application prospects.

Description

(1) Technical field [0001] The present invention relates to a nucleoside-diphosphate kinase (nucleoside-diphosphate kinase) derived from the "Baling" producing strain Cordyceps sinensis, which is involved in the synthesis and metabolism of pyrimidine nucleoside triphosphates (deoxy) pyrimidine nucleoside. The gene encoding this enzyme and its application. (2) Background technology [0002] Cordyceps sinensis (Berk.) Sacc. is a complex of Cordyceps sinensis (Berk.) Sacc. parasitic on Lepidoptera Hepialus armoricanus Oberthur larvae and larvae corpses ). Cordyceps sinensis is a kind of cherished traditional fungal medicinal resource. It has the characteristics of diverse metabolites and biological activities, and has shown great application and development prospects in the field of biomedicine. Cordyceps has attracted much attention for its wide and obvious medicinal effects, and is highly respected worldwide. Chinese medicine believes that Cordyceps enters the lung and kidney t...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N1/21C12P19/30C12R1/19
CPCY02P20/52
Inventor 郑裕国柳志强吴晖李邦良吴玲芳许静许峰薛亚平袁水金王鸿艳
Owner ZHEJIANG UNIV OF TECH
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