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Enzyme for synthesizing and metabolizing inosine monophosphate of Cordyceps sinensis(Berk.)Sacc. Hirsutella sinensis and application thereof

A technology of hypoxanthine nucleotide and Trichosporum sinensis, applied in the field of AMP deaminase, can solve the problems of rare research on genes and proteins, achieve great application prospects, expand production, and achieve high expression effects

Active Publication Date: 2012-09-26
ZHEJIANG UNIV OF TECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, the purine nucleoside producing bacteria used are mainly Bacillus subtilis, and Cordyceps sinensis, which is an important anabolic purine nucleoside, only stays in the analysis of metabolite components and efficacy research. Studies on related genes and proteins in the nucleoside synthetic metabolic pathway are still rare

Method used

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  • Enzyme for synthesizing and metabolizing inosine monophosphate of Cordyceps sinensis(Berk.)Sacc. Hirsutella sinensis and application thereof
  • Enzyme for synthesizing and metabolizing inosine monophosphate of Cordyceps sinensis(Berk.)Sacc. Hirsutella sinensis and application thereof
  • Enzyme for synthesizing and metabolizing inosine monophosphate of Cordyceps sinensis(Berk.)Sacc. Hirsutella sinensis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Cultivation of "Bailing" production fungus Cordyceps sinensis

[0028] Source of the strain: Firstly, 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 Objects Collection Center, the deposit number is CCTCC No: M 2011278, which has been disclosed in the previous patent application CN102373190A.

[0029] Inoculate the strain on the slant, and the medium formula (this is the liquid formula before solidification, prepared according to the following proportions before making the slant) 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...

Embodiment 2

[0030] Example 2: Extraction of total RNA of "Bailing" production fungus Cordyceps sinensis

[0031]The total RNA was extracted with TRIzol reagent, and the steps were 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 powdery, and divide 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 0.2mL chloroform, shake vigorously for 15s, let stand on ice for 2~3min, centrifuge at 12000rpm at 4°C for 15min, separate the layers, and take the upper aqueous phase, about 600μL. 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 pellet, let stand on ice for 10 min, centrifuge at 4°C, 7500 rpm for 15 min...

Embodiment 3

[0032] Example 3: Sequencing of the RNA sample of "Bailing" production fungus Cordyceps sinensis

[0033] 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 QiaQuickPCR reagent After the cassette was purified and eluted with EB buffer, end repair was performed, polyA was added and sequencing adapters were connected, and then agarose gel electrophoresis was used for fragment size selection, and finally PCR amplification was performed. The built sequencing library was 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 sequenci...

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Abstract

The invention relates to an adenosine monophosphate (AMP) adenosine deaminase for synthesizing and metabolizing an inosine monophosphate from adenine nucleotide of Cordyceps sinensis(Berk.)Sacc. Hirsutella sinensis from 'Bailing' production strain, a gene coding the enzyme and application thereof. The AMP adenosine deaminase comprises the proteins shown in SEQ ID No. 1 and SEQ ID No. 2, and the coding gene of the AMP adenosine deaminase corresponds to the nucleotide sequence shown in SEQ ID No. 3 and SEQ ID No.4. According to the invention, the metabolizing way for synthesizing the inosine monophosphate from the adenine nucleotide is researched in detail from the principle, and the cloned DNA containing the nucleotide sequence provided by the invention can be transferred to an engineering strain by transduction, transformation, and combined transfer; the expression of the gene is biologically synthesized by regulating the inosine monophosphate, the host inosine monophosphate is endowed with high expressivity, an effective way is provided for increasing the yield of the inosine monophosphate, and the invention has a great application prospect.

Description

(1) Technical field [0001] The present invention relates to a group of AMP deaminases (adenosine deaminase) involved in the synthesis and metabolism of hypoxanthine nucleotides from adenine nucleotides from the "Bailing" production fungus Cordyceps sinensis, the gene encoding this enzyme and its application. (2) Background technology [0002] 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 belie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/14C12N15/55C12N1/15C12N1/19C12N1/21C12P19/32
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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