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Cordycepin anabolism-related enzyme from cordyceps sinensis hirsutella sinensis, gene and application

A technology of Trichospora and Cordyceps sinensis in China, applied in application, genetic engineering, plant genetic improvement and other directions, can solve rare problems and achieve the effect of expanding yield, high expression, and major application prospects

Active Publication Date: 2013-01-30
ZHEJIANG UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are few reports on the enzymes involved in the synthesis and metabolism of cordycepin

Method used

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  • Cordycepin anabolism-related enzyme from cordyceps sinensis hirsutella sinensis, gene and application
  • Cordycepin anabolism-related enzyme from cordyceps sinensis hirsutella sinensis, gene and application
  • Cordycepin anabolism-related enzyme from cordyceps sinensis hirsutella sinensis, gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Cultivation of "Bailing" production fungus Cordyceps sinensis

[0060] The source of the strain: Firstly, the natural Cordyceps sinensis was collected from Qinghai, and brought back to Hangzhou for isolation and screening, and the L0106 strain was obtained, which was identified as Hirsrtella Sinensis and named as Hirsrtella Sinensis ( Hirsrtella Sinensis L0106), this strain is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC No: M 2011278, and the preservation date is August 5, 2011.

[0061] The bacterial classification is inoculated on the slant, and the culture medium formula (this is the liquid formula before solidification, and the slant is 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 ...

Embodiment 2

[0062] Example 2: Extraction of total RNA of "Bailing" production fungus Cordyceps sinensis

[0063] Use TRIzol reagent to extract total RNA. The steps are as follows: 1) Grinding with liquid nitrogen: Take 1 g of fresh bacteria and put it into a mortar, add liquid nitrogen repeatedly to grind until it becomes powdery, divide it into a pre-cooled 1.5mL centrifuge tube, add 1mL TRIzol reagent, mix well, and let stand on ice for 5min to completely separate the nucleic acid-protein complex. 2) RNA isolation: Add 0.2 mL of chloroform, shake vigorously for 15 s, let stand on ice for 2-3 min, centrifuge at 4°C, 12000 rpm for 15 min, separate the layers, and take the upper aqueous phase, about 600 μL. 3) RNA precipitation: add 500 μL of isopropanol, let stand on ice for 10 minutes, centrifuge at 12000 rpm at 4°C for 10 minutes, and discard the supernatant. 4) RNA washing: add 1 mL of 75% (v / v) ethanol, suspend the precipitate, let stand on ice for 10 min, centrifuge at 4°C, 7500 rpm...

Embodiment 3

[0064] Example 3: Sequencing of the RNA sample of "Bailing" production fungus Cordyceps sinensis

[0065] After extracting the total RNA from the sample, the mRNA was enriched with Oligo(dT) magnetic beads. Add fragmentation buffer to break mRNA into short fragments (200-700bp), use mRNA as a template, use six-base random primers (random hexamers) to synthesize the first cDNA strand, then synthesize the second cDNA strand, and then pass through QiaQuick PCR After the kit is purified and eluted with EB buffer, end repair is performed, polyA is added and sequencing adapters are connected, and then agarose gel electrophoresis is used for fragment size selection, and finally PCR amplification is performed, and the built sequencing library is sequenced with Illumina GAIIx . The original image data obtained by sequencing is converted into sequence data through base calling, that is, raw data or raw reads. The reads containing only the adapter sequence in the original sequencing re...

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Abstract

The present invention provides a set of cordycepin anabolism-related enzymes from bering producing strain cordyceps sinensis hirsutella sinensis-participated ADP ribose starting, genes encoding the enzymes, and application thereof. The related enzymes include proteins shown as SEQ ID No. 1-17, and the coding genes correspond to nucleotide sequences shown as SEQ ID No. 18-34. The invention conducts detailed researches on the principle of metabolic pathways of cordycepin synthesis from bering producing strain cordyceps sinensis hirsutella sinensis ADP ribose. The cloned DNA of the nucleotide sequences provided by the invention can be transferred into engineering bacteria by a transduction, transformation and conjugal transfer method. By adjusting the expression of cordycepin biosynthesis genes, high expression of cordycepin for the host is given.

Description

(1) Technical field [0001] The invention relates to a group of related enzymes from the "Bailing" production fungus Cordyceps sinensis that participate in the synthesis and metabolism of cordycepin from ADP ribose, genes encoding these enzymes and applications thereof. (2) Background technology [0002] Cordyceps sinensis (Cordyceps sinensis (Berk.) Sacc.) is a complex of Cordyceps sinensis parasitizing on the larvae of Lepidoptera (Lepidoptera) bat moth (Hepialus armoricanus Oberthur) larvae and larval corpses (including subunits and worm bodies ). Cordyceps sinensis is a kind of precious traditional fungal medicinal resources, which has the characteristics of diverse metabolites and biological activities, and has shown great application and development prospects in the field of biomedicine. Cordyceps sinensis has attracted much attention for its extensive and obvious medicinal effects, and is highly respected all over the world. Chinese medicine believes that Cordyceps s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N9/10C12N9/00C12N9/88C12N9/22C12P19/40C12N15/54C12N15/52C12N15/60C12N15/55C12N15/70C12N1/21C12R1/465
Inventor 郑裕国李邦良吴晖柳志强许静陈丽芳许峰薛亚平袁水金王鸿艳
Owner ZHEJIANG UNIV OF TECH
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