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Fructokinase from fungal traditional Chinese medicine and coding gene and use thereof

A fructokinase, encoding gene technology, applied in the field of genetic engineering, to achieve the effects of expanding production, major application prospects, and high expression

Active Publication Date: 2021-09-24
北京中医药大学深圳医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few studies on related genes and proteins in its biosynthetic metabolic pathway.

Method used

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  • Fructokinase from fungal traditional Chinese medicine and coding gene and use thereof
  • Fructokinase from fungal traditional Chinese medicine and coding gene and use thereof
  • Fructokinase from fungal traditional Chinese medicine and coding gene and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The extraction of the total RNA of the fungal traditional Chinese medicine Trichosanthes sinensis of embodiment 1

[0039] Use TRIzol reagent to extract the total RNA of the fungal traditional Chinese medicine Trichosporum sinensis. The specific steps are: ① Liquid nitrogen grinding: add liquid nitrogen repeatedly to grind 1 g of fresh bacteria to a powdery state, divide them into pre-cooled 1.5mL centrifuge tubes, add Mix 1mL TRIzol reagent evenly, and let stand on ice for 5min to completely separate the nucleic acid-protein complex. ② RNA isolation: After adding 0.2mL chloroform, vigorously shake and mix for 15s, let stand on ice for 2-3min, centrifuge at 12000rpm, 4°C for 15min, separate layers, and take the upper aqueous phase. ③ RNA precipitation: After adding 500 μL of isopropanol, let stand on ice for 10 minutes, centrifuge at 12,000 rpm and 4°C for 10 minutes, and discard the supernatant. ④ RNA washing: Add 1 mL of 75% (v / v) ethanol, suspend the pellet, let it ...

Embodiment 2

[0040] Example 2 The sequencing of fungal traditional Chinese medicine Trichosinus sinensis RNA sample

[0041] After extracting the total RNA from the sample, the mRNA was enriched with Oligo(dT) magnetic beads. Add fragmentation buffer to fragment mRNA into short fragments (150-700bp), use mRNA as a template, use six-base random primers to synthesize the first cDNA strand, and then synthesize the second cDNA strand, then purify and add to the QiaQuick PCR kit After elution 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, and the built sequencing library was sequenced with Illumina GA IIx.

Embodiment 3

[0042] Example 3 Assembly of fungal traditional Chinese medicine Mortierella sinensis RNA short-read sequence

[0043] Use the short reads assembly software SOAPdenovo (Li, Zhu et al. De novo assembly of human genomes with massively parallel short read sequencing [J]. Genome Res, 2010, 20:265-272.) to do de novo transcriptome assembly. Use paired-end reads to fill holes in Scaffold, and finally get a Unigene sequence that contains the least N and cannot be extended at both ends. Secondly, the Unigene sequence was compared with the protein database Swiss-Prot, nr, COG and KEGG by blastx (evalue<0.00001), and the protein with the best alignment result was used to determine the sequence direction of the Unigene. If there are conflicts in the alignment results between different libraries, the sequence direction of Unigene is determined according to the priority of nr, Swiss-Prot, KEGG and COG.

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Abstract

The invention discloses fructokinase from a fungal traditional Chinese medicine and a coding gene and use thereof. The fungal traditional Chinese medicine fructokinase has an amino acid sequence as shown in SEQ ID NO: 1 and a nucleotide sequence of the coding gene as shown in SEQ ID NO: 2. The fungal traditional Chinese medicine fructokinase participates in synthesis of main precursor monosaccharide D-fructose of cordyceps polysaccharide from D-fructose-6-phosphoric acid. A fungal traditional Chinese medicine hirsutella sinensis polysaccharide biosynthetic metabolism pathway is studied in details in principle. A cloned DNA containing the nucleotide sequence can be transferred into engineering bacteria by transduction, transformation and conjugal transfer methods. An expression of a D-fructose biosynthetic gene is regulated, the host is endowed with high expressivity of D-fructose, an effective way is provided for increasing the yield of the D-fructose and a derivative thereof, and the fructokinase has a great application prospect.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering, more specifically, relates to a kind of fructokinase from fungal traditional Chinese medicine and its encoding gene and application. Background technique [0002] Traditional Chinese medicine is a treasure of the Chinese nation, a unique health resource of our country, and an important part of the world's traditional medicine. In 2015, Professor Tu Youyou won the "Nobel Prize in Physiology or Medicine" for her outstanding contribution in the research of artemisinin in the treatment of malaria. Traditional Chinese medicine has played a unique role in modern medicine and has attracted widespread attention in the world. [0003] Poria cocos, Cordyceps sinensis (Herba sinensis), silkworm, shiitake mushrooms, fungus and cicadae, etc. are traditional fungal Chinese medicinal materials in my country, and polysaccharides, terpenes, nucleosides, sterols and other functional active comp...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/70C12N1/21C12P19/02C12R1/19
CPCC12N9/1205C12N15/70C12P19/02C12Y207/01004Y02A50/30
Inventor 林善吴芬芳
Owner 北京中医药大学深圳医院
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