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Cordyceps sinensis delta-5-desaturase used in anabolism of eicosapentaenoic acid, and gene and application thereof

An eicosapentaene coenzyme and synthetic metabolism technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of rare research on genes and proteins, achieve great application prospects, expand production, and high expression Effect

Active Publication Date: 2013-03-20
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 unsaturated fatty acid producing bacteria used are mainly Bacillus subtilis, and Cordyceps sinensis, which is an important synthetic and metabolic unsaturated fatty acid, still only stays in the analysis of metabolite components and efficacy research, and the synthesis of unsaturated fatty acids by Cordyceps sinensis Studies on related genes and proteins in metabolic pathways are still rare

Method used

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  • Cordyceps sinensis delta-5-desaturase used in anabolism of eicosapentaenoic acid, and gene and application thereof
  • Cordyceps sinensis delta-5-desaturase used in anabolism of eicosapentaenoic acid, and gene and application thereof
  • Cordyceps sinensis delta-5-desaturase used in anabolism of eicosapentaenoic acid, and 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, 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] 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 1.0%, and w...

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 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 mi...

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 delta-5-desaturase from a Corbrin-production bacterium cordyceps sinensis hirsutella sinensis. The delta-5-desaturase participates in eicosapentaenoic coenzyme A anabolism started from eicosatetraenoic coenzyme A. The invention also relates to a gene coding the enzyme, and an application thereof. The amino acid sequence of the delta-5-desaturase has more than 90% homology with a sequence represented by SEQ ID No.1 or SEQ ID No.3. From the principle, the invention carries out detailed researches upon the metabolic pathway of eicosatetraenoic coenzyme A to synthesizing the eicosapentaenoic coenzyme A. A cloned DNA comprising the nucleotide sequence provided by the invention can be transferred to engineering bacteria through methods of transduction, transformation, and combined transferring. Through the regulation upon eicosapentaenoic coenzyme A biosynthetic gene expression, high expression of a host delta-5-desaturase is provided, an effective approach is provided for increasing eicosapentaenoic acid yield. The application has an important application prospect.

Description

(1) Technical field [0001] The invention relates to a △-5-dehydrogenase (△-5 desaturase) involved in the synthesis and metabolism of eicosapentaene-coenzyme A from the "Bailing" production fungus Cordyceps sinensis. , the gene encoding this enzyme and its application. Eicosapentaene-CoA is then hydrolyzed to generate eicosapentaenoic acid. (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 t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N1/15C12N1/19C12N1/21C12P19/32
Inventor 郑裕国柳志强吴晖李邦良许静林善许峰薛亚平袁水金王鸿艳
Owner ZHEJIANG UNIV OF TECH
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