Cordyceps Chinese Hirsutella carbamyl phosphate synthetase, coding gene and application thereof

A carbamoyl phosphate, Trichospora chinensis technology, applied in the directions of enzymes, microorganism-based methods, enzymes, etc., can solve the problems of rare gene and protein research, and achieve the effect of major application prospects, expansion of production, and high expression.

Active Publication Date: 2014-07-02
ZHEJIANG UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, 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 related genes and proteins in the synthesis and metabolism pathway of pyrimidine nucleosides of Cordyceps sinensis is still rare

Method used

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  • Cordyceps Chinese Hirsutella carbamyl phosphate synthetase, coding gene and application thereof
  • Cordyceps Chinese Hirsutella carbamyl phosphate synthetase, coding gene and application thereof
  • Cordyceps Chinese Hirsutella carbamyl phosphate 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 Object Depository Center, the deposit number is CCTCCNo: M 2011278, which has been disclosed in the previously applied patent CN102373190A.

[0036] The strain 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 phosphate 0.05% ...

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: Take 1 g of fresh wet bacteria (prepared in Example 1) and put it into a mortar, add liquid nitrogen repeatedly to fully grind to powder, and then pack it into pre- Add 1mL TRIzol reagent to a cold 1.5mL centrifuge tube, 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 12,000 rpm at 4°C for 15 min, separate 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 ...

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 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. The built sequencing library is carried out with Illumina GA IIx sequencing. 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 sequ...

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Abstract

The invention relates to a carbamyl phosphate synthetase from Bailing production bacterium Cordyceps Chinese Hirsutella for synthesizing metabolic carbamyl phosphate from L-glutamic acid, a coding gene and application thereof. The amino acid sequence of the carbamyl phosphate synthetase has more than 90% of homology with sequence disclosed as SEQ ID NO.1; and the nucleotide sequence of the coding gene has more than 90% of homology with sequence disclosed as SEQ ID NO.2. The invention researches the metabolic pathway of L-glutamic acid synthesized carbamyl phosphate synthetase 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 carbamyl phosphate biosynthesized gene, the host carbamyl phosphate is endowed with high expressivity, thereby providing an effective way for enhancing the yield of the carbamyl phosphate and having great application prospects.

Description

(1) Technical field [0001] The invention relates to a carbamoyl-phosphate synthase (carbamoyl-phosphate) derived from the production fungus Cordyceps sinensis of "Bai Ling" which participates in the first step of the three-step reaction of L-glutamic acid synthesis and metabolism of dihydroorotic acid. synthase, CPS), the gene encoding the enzyme and its application. (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 medic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N15/52C12N1/21C12P13/00C12R1/19
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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