Enzyme synthesized by Chinese caterpillar fungus hirsutella sinensis to metabolize guanine nucleotide and genes and application of enzyme

A Chinese hairpin and synthetic metabolism technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of rare research on genes and proteins, achieve great application prospects, expand production, and achieve high expression effects

Inactive Publication Date: 2012-09-26
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, the purine nucleoside producing bacteria used are mainly Bacillus subtilis, and Cordyceps sinensis, which is an important anabolic purine nucleoside, on

Method used

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  • Enzyme synthesized by Chinese caterpillar fungus hirsutella sinensis to metabolize guanine nucleotide and genes and application of enzyme
  • Enzyme synthesized by Chinese caterpillar fungus hirsutella sinensis to metabolize guanine nucleotide and genes and application of enzyme
  • Enzyme synthesized by Chinese caterpillar fungus hirsutella sinensis to metabolize guanine nucleotide and genes and application of enzyme

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 Cartridges were purified and eluted with EB buffer, followed by end repair, polyA addition and sequencing adapters, and then agarose gel electrophoresis for fragment size selection, and finally PCR amplification, 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 removed for subsequent anal...

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Abstract

The invention relates to IMP (inosine monophosphate) dehydrogenase synthesized by 'Bailing' producing fungus Chinese caterpillar fungus hirsutella sinensis to metabolize guanine nucleotide based on xanthosine monophosphate and coding genes and application of the IMP dehydrogenase. The IMP dehydrogenase comprises proteins shown by SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, and coding genes of the IMP dehydrogenase correspond to nucleotide sequences shown by SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6. Detailed studies on metabolic pathways of the xanthosine monophosphate synthesizing the guanine nucleotide in principle are carried out and include that cloned DNAs (deoxyribonucleic acids) in the provided nucleotide sequences can be transferred into engineering bacteria by means of transduction, transformation and transconjunction, and high expressivity is given to the host guanine nucleotide by adjusting expressions of guanine nucleotide biosynthetic genes, so that an effective way for increasing output of the guanine nucleotide is provided, and the IMP dehydrogenase and the coding genes have significant application prospects.

Description

(1) Technical field [0001] The invention relates to a GMP synthetase from the "Bailing" production fungus Cordyceps sinensis and participates in the synthesis and metabolism of guanine nucleotides from xanthine nucleotides, the gene encoding the 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 moths (Hepialus armoricanus Oberthur) and the larvae corpse (including the subunit and the body ). 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 t...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N1/15C12N1/19C12N1/21C12P19/32
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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