Relevant enzymes for preparing mannitol by performing anabolism on Chinese caterpillar fungus and hirsutella sinensis, gene and application thereof

A Chinese technology of Trichosporum and Cordyceps sinensis, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problem of rare research on the biosynthesis and metabolism of mannitol, and achieve the effect of high expression

Active Publication Date: 2012-03-14
ZHEJIANG UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are more and more studies on the production of mannitol through microbial fermentation pathways, and some research progress has been made, but the research on the biosynthesis and metabolism of mannitol is still rare, especially on the synthetic metabolism of mannitol in Cordyceps sinensis Alcohol research also focuses on the optimization of fermentation conditions

Method used

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  • Relevant enzymes for preparing mannitol by performing anabolism on Chinese caterpillar fungus and hirsutella sinensis, gene and application thereof
  • Relevant enzymes for preparing mannitol by performing anabolism on Chinese caterpillar fungus and hirsutella sinensis, gene and application thereof
  • Relevant enzymes for preparing mannitol by performing anabolism on Chinese caterpillar fungus and hirsutella sinensis, gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Cultivation of "Bailing" production fungus Cordyceps sinensis

[0034] Source of the strain: firstly collect the natural Cordyceps sinensis from Qinghai, and bring it back to Hangzhou for isolation and screening, and obtain the L0106 strain, which is identified as Hirsrtella Sinensis by strain identification. This strain is preserved in a typical culture in China Collection Center, address: China, Wuhan, Wuhan University, 430072, deposit number is CCTCCNo: M 2011278, deposit date: August 5, 2011.

[0035] The bacterial classification 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 dihyd...

Embodiment 2

[0036] Example 2: Extraction of total RNA of "Bailing" production fungus Cordyceps sinensis

[0037] The total RNA was extracted with TRIzol reagent, and the steps were as follows: 1) Grinding with liquid nitrogen: Take 1 g of wet bacteria and put it into a mortar, add liquid nitrogen repeatedly and grind until powdery, then pack it into a pre-cooled 1.5mL centrifuge tube, 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 0.2 mL of chloroform, shake vigorously for 15 s, let stand on ice for 2-3 min, centrifuge at 4°C, 12000 rpm for 15 min, separate the 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% ethanol, suspend the precipitate, let stand on ice for 10 min, centrifuge at 4°C, 7500 rpm for...

Embodiment 3

[0038] 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, and the built sequencing library is 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 sequencing reads were removed for subsequent analysis.

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Abstract

The invention provides a group of relevant enzymes for preparing mannitol by performing anabolism on Chinese caterpillar fungus serving as a multifunctional production fungus and hirsutella sinensis based on glucose, a gene for encoding these enzymes and application thereof. The relevant enzymes include (1) hexokinase: manA1-A6 proteins of which the sequences are SEQ ID No.1-6, (2) phosphoglucoisomerase: manB1-B3 proteins of which the sequences are SEQ ID No.7-9, and (3) mannitol-1-P dehydrogenase: manC protein of which the sequence is SEQ ID No.10. In the invention, detailed researches are performed on the metabolic pathway of mannitol synthesized by using Chinese caterpillar fungus serving as a multifunctional production fungus, hirsutella sinensis and glucose on the aspect of principle, cloned DNA (Deoxyribose Nucleic Acid) comprising a nucleotide sequence provided by the invention can be transferred into engineering bacteria with transduction, conversion and conjugal transfer methods, and host mannitol is endowed with high expression by regulating the expression of a biosynthetic gene of the mannitol.

Description

(1) Technical field [0001] The invention relates to a group of related enzymes from the "Bailing" production fungus Cordyceps sinensis Mortierella sinensis involved in the synthesis and metabolism of mannitol from glucose, genes encoding these enzymes and applications thereof. (2) Background technology [0002] Cordyceps sinensis (Berk.) Sacc., also known as Cordyceps sinensis and Cordyceps sinensis, is a complex of sub-seats and larval corpses that parasitize on the larvae of the larvae of the family Mothidae. 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. Scholars at home and abroad have done a lot of work in the investigation of Cordyc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N9/92C12N9/04C12P7/18C12N15/54C12N15/61C12N15/53C12N15/70C12N1/14C12R1/645C12R1/19
Inventor 郑裕国李邦良吴晖柳志强许静袁水金许峰陈丽芳薛亚平沈寅初
Owner ZHEJIANG UNIV OF TECH
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