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Cordyceps sinensis ctp synthetase, coding gene and application thereof

A technology of Cordyceps sinensis and synthase, applied in the field of CTP synthase, can solve the problems of rare gene and protein research, and achieve the effect of major application prospect, high expression, and yield expansion

Active Publication Date: 2017-06-13
ZHEJIANG UNIV OF TECH +2
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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 ctp synthetase, coding gene and application thereof
  • Cordyceps sinensis ctp synthetase, coding gene and application thereof
  • Cordyceps sinensis ctp synthetase, coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0035] 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, and the strain was identified as Hirsutella Sinensis, which was preserved in a typical culture in China Depository Center, the deposit number is CCTCC No: M 2011278, which has been disclosed in the previously applied patent CN102373190A.

[0036] 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 sulfate 0.05%, potassium dihydrogen phos...

Embodiment 2

[0037] Example 2: Extraction of total RNA of "Bailing" production fungus Cordyceps sinensis

[0038] The total RNA was extracted with TRIzol reagent, and the steps were as follows: 1) Grinding with liquid nitrogen: put 1 g of fresh bacteria into a mortar, add liquid nitrogen repeatedly to grind until powdery, and distribute it into pre-cooled 1.5mL centrifuge tubes. 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 fo...

Embodiment 3

[0039] Example 3: Sequencing of the RNA sample of "Bailing" production fungus Cordyceps sinensis

[0040] 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 the QiaQuick PCR kit After purification and elution with EB buffer, 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, and the built sequencing library was sequenced with Illumina GA IIx. 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 reads were re...

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Abstract

The invention relates to CTP (Cytidine Triphosphate) synthetase from Bailing producing strain cordyceps sinensis hirsutella sinensis participated in synthesis of metabolic cytidine triphosphate from uridine triphosphate, a gene for coding the synthetase and application of the CTP synthetase; the CTP synthetase has an amino acid sequence represented by SEQ ID No.2 and with the homology of more than 90%; the coding gene has a nucleotide sequence represented by SEQ ID No.4 and with the homology of more than 90%; a metabolic pathway of synthesis of cytidine triphosphate by uridine triphosphate is researched in a detailed manner in principle; cloned DNA of the nucleotide sequence provided by the invention can be transferred into engineering bacteria through transduction, conversion and conjugal transfer methods; by adjusting expression of a biosynthetic gene of cytidine triphosphate, the high expressivity of host cytidine triphosphate is given; an effective way is provided for increasing the yield of cytidine triphosphate; and therefore, the CTP synthetase has great application prospect.

Description

[0001] (1) Technical field [0002] The invention relates to a CTP synthase (CTP synthase) involved in the synthesis and metabolism of cytidine triphosphate derived from uridine triphosphate, a gene encoding the enzyme and its application. [0003] (2) Background technology [0004] 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 sinensis enters the two m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N15/52C12P19/30C12N15/70C12N1/21C12R1/19
CPCC12N9/93C12P19/305C12Y603/04002
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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