Hirsutella sinensis ketohexokinasee, coding gene thereof and application of hirsutella sinensis ketohexokinasee and coding gene thereof

A technology of chrysohexulose and coding genes, which is applied in the field of genetic engineering to achieve the effect of expanding production and high expression

Active Publication Date: 2022-01-07
北京中医药大学深圳医院
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AI Technical Summary

Benefits of technology

This patented technology allows for efficient use of certain enzyme called xylitol dehydrogenases (XLDs) that are found naturally on plants like rice grains. These XLDS have been identified through their ability to catalyze chemical reactions between glucoses and other sugars. They also contain specific proteins involved in carbolelic polymerization processes during cell growth. By controllably modifying these genes overexpressing them, researchers could creatively produce various types of compounds containing this type of molecule.

Problems solved by technology

This patented technical problem addressed by these inventors relates to extracting and purifying certain types of polyglycosylated carbohydrates that can be useful in treatments or prevention against cancer, inflammations, oxidative stress, cell deaths caused by radiation therapy, chemotherapies, hormones, antioxic agents, cytokines, etc., particularly when they come into contact with living cells containing enzymatic receptors involved in various biochemistry processes like signal transducers and growth factors.

Method used

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  • Hirsutella sinensis ketohexokinasee, coding gene thereof and application of hirsutella sinensis ketohexokinasee and coding gene thereof
  • Hirsutella sinensis ketohexokinasee, coding gene thereof and application of hirsutella sinensis ketohexokinasee and coding gene thereof
  • Hirsutella sinensis ketohexokinasee, coding gene thereof and application of hirsutella sinensis ketohexokinasee and coding gene thereof

Examples

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Comparison scheme
Effect test

Embodiment 1

[0036] The extraction of the total RNA of the fungal traditional Chinese medicine Trichosanthes sinensis of embodiment 1

[0037]The total RNA of the fungal Chinese medicine Mortierella sinensis was extracted using TRIzol reagent, and the specific experimental steps were as follows: (1) Grinding with liquid nitrogen: 1 g of fresh bacteria was repeatedly added with liquid nitrogen, and fully ground to powder, and then packed into In a pre-cooled 1.5mL centrifuge tube, mix with 1mL TRIzol, and let stand on ice for 5min to completely separate the protein-nucleic acid complex. (2) RNA isolation: add 0.2mL chloroform to the tube, vortex and mix for 15s, let stand on ice for 2-3min, centrifuge at 4°C and 12000rpm for 15min, separate the layers and take the upper aqueous phase. (3) RNA precipitation: After adding 500 μL of isopropanol to the tube, let it stand on ice for 10 minutes, centrifuge at 4°C and 12,000 rpm for 10 minutes, and discard the supernatant. (4) Washing of RNA: Add...

Embodiment 2

[0038] Example 2 The sequencing of fungal traditional Chinese medicine Trichosinus sinensis RNA sample

[0039] First, after extracting the total RNA of the fungal traditional Chinese medicine sample, its mRNA was enriched with Oligo(dT) magnetic beads. And add fragmentation buffer to break the mRNA into short fragments (150-700bp), use the mRNA as a template, use six-base random primers to synthesize the first cDNA strand, and then synthesize the second cDNA strand, which is passed through the QiaQuick PCR reagent After the cassette is purified, add EB buffer to elute, perform end repair, add polyA and connect the sequencing adapter, then use agarose gel electrophoresis to select the size of the fragment, and finally perform PCR amplification. The built sequencing library is carried out by Illumina GA IIx sequencing.

Embodiment 3

[0040] Example 3 Assembly of short-read RNA sequences of the fungal traditional Chinese medicine Mortierella sinensis and functional annotation of Unigene

[0041] First, use the SOAPdenovo short reads assembly software (Li, Zhu et al. De novo assembly of human genomes with massively parallel short read sequencing [J]. Genome Res, 2010, 20:265-272.) to de novo assemble the transcriptome. Secondly, use the paired-end reads method to fill holes in the Scaffold, and finally get a Unigene sequence that cannot be extended at both ends and contains the least N. Secondly, use the protein database Swiss-Prot, nr, COG and KEGG to perform blastx alignment on the Unigene sequence (evalue<0.00001), and use the protein with the best alignment result to determine the sequence direction of the Unigene. If there are conflicts in the comparison results between different databases, the sequence direction of Unigene is determined according to the priority of nr, Swiss-Prot, KEGG and COG.

[004...

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Abstract

The invention discloses ketohexokinasee from fungus traditional Chinese medicine hirsutella sinensis, a coding gene of the ketohexokinasee and application of the ketohexokinasee and coding gene of the ketohexokinasee. The amino acid sequence of the hirsutella sinensis ketohexokinasee is shown as SEQ ID NO: 1, and the nucleotide sequence of the coding gene of the hirsutella sinensis ketohexokinasee is shown as SEQ ID NO: 2. The hirsutella sinensis ketohexokinasee participates in synthesis of main precursor monosaccharide D-fructose of cordyceps polysaccharide from D-fructose-1-phosphoric acid. Based on the hirsutella sinensis ketohexokinasee, through a genetic engineering means, expression of the biosynthetic gene of the D-fructose is regulated, the host D-fructose is endowed with high expressivity, and an effective approach is provided for enlarging the yield of the D-fructose and a derivative thereof.

Description

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Claims

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Application Information

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Owner 北京中医药大学深圳医院
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