Enzyme for synthesizing and metabolizing adenine of Cordyceps sinensis(Berk.)Sacc. Hirsutella sinensis, and gene and application of enzyme

A Chinese Trichospora, anabolic technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problem of rare gene and protein research, and achieve the effect of major application prospects, expansion of production, and high expression.

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,...

Method used

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  • Enzyme for synthesizing and metabolizing adenine of Cordyceps sinensis(Berk.)Sacc. Hirsutella sinensis, and gene and application of enzyme
  • Enzyme for synthesizing and metabolizing adenine of Cordyceps sinensis(Berk.)Sacc. Hirsutella sinensis, and gene and application of enzyme
  • Enzyme for synthesizing and metabolizing adenine of Cordyceps sinensis(Berk.)Sacc. Hirsutella sinensis, and gene 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 Object Collection Center, the preservation number is CCTCC No: M 2011278, which has been disclosed in the prior 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 ...

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 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 an adenine phosphoribosyltransferase for synthesizing and metabolizing an adenine from adenine nucleotide of Cordyceps sinensis(Berk.)Sacc. Hirsutella sinensis from 'Bailing' production strain, a gene coding the enzyme and application of the adenine phosphoribosyltransferase. The amino acid sequence of the adenine phosphoribosyltransferase is shown in SEQ ID No. 1, and the coding gene of the adenine phosphoribosyltransferase corresponds to the nucleotide sequence shown in SEQ ID No. 2. According to the invention, the metabolizing way for synthesizing the adenine 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; through regulating the gene expression of the adenine biological synthesis, the host adenine is endowed with high expressivity, an effective way is provided for increasing the yield of the adenine, and the invention has a great application prospect.

Description

(1) Technical field [0001] The present invention relates to an adenine phosphoribosyltransferase (adenine phosphoribosyltransferase; APRT) involved in the synthesis and metabolism of adenine from adenine nucleotides from the "Bailing" production fungus Cordyceps sinensis, 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 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 m...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N1/15C12N1/19C12N1/21C12P17/18
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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