Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A kind of uridine-5'-diphosphate xylose synthase derived from Dieffenbachia tiger eye, its nucleotide sequence and application

A technology of xylose diphosphate and synthetase, applied in the field of genetic engineering

Active Publication Date: 2021-09-14
INST OF MATERIA MEDICA CHINESE ACAD OF MEDICAL SCI
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] So far, UDP-xylose synthase has been found and cloned in plants such as Arabidopsis thaliana, Hordeum vulgare, and bacteria such as Sinorhizobium meliloti gene, but there is no report on the isolation of this protein from the Asparagus plant Ornithogalumcaudatum

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of uridine-5'-diphosphate xylose synthase derived from Dieffenbachia tiger eye, its nucleotide sequence and application
  • A kind of uridine-5'-diphosphate xylose synthase derived from Dieffenbachia tiger eye, its nucleotide sequence and application
  • A kind of uridine-5'-diphosphate xylose synthase derived from Dieffenbachia tiger eye, its nucleotide sequence and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Transcriptome Sequencing and Sequence Analysis of Dieffenbachia tiger-eye

[0032] After extracting the total RNA from the sterile bulb of Dieffenbachia tiger eye, using the mRNA as a template, the first cDNA strand was synthesized with six base random primers (randomhexamers), and then the second cDNA was synthesized by adding buffer, dNTPs, RNase H and DNA polymerase I After purification by QiaQuick PCR kit and elution with EB buffer, end repair, poly(A) was added and sequencing adapters were connected, then fragment size selection was performed by agarose gel electrophoresis, and finally PCR amplification was carried out to construct Good sequencing library with Illumina HiSeq TM 2000 for sequencing.

[0033] The original image data obtained by sequencing is converted into sequence data through base calling, that is, raw data or rawreads. Perform data filtering on raw reads, remove reads with adapters, duplicates, and low-quality sequencing, and obtain c...

Embodiment 2

[0035] Example 2 Cloning of OsaUXS1 gene

[0036]Take 100mg of aseptic bulbs of Dieffenbachia tiger's eye, freeze them quickly in liquid nitrogen, grind them into fine powder with a mortar, and use Trizol extraction to extract total RNA from the aseptic bulbs of Dieffenbachia tiger's eye. Using RT-PCR Kit (ReverTra-Plus-, TOYOBO company) to reverse transcribe the total RNA of Diofenbachia sterile bulbs into cDNA. The reverse transcription system and procedure are as follows: add 1 μg of total RNA to 20 μL system, RNase Free H 2 O 4 μL, Oligo(dT) 20 1 μL, after incubating at 65°C for 5 minutes, immediately place it on ice to cool, then add 4 μL of 5×RT buffer, 1 μL of RNase Inhibitor (10U / μL), 1 μL of dNTP Mixture (10mM) and ReverTra Ace to the above tube, at 30°C Incubate for 10 minutes, incubate at 42°C for 60 minutes, denature at 85°C for 5 minutes, and place on ice for 5 minutes to complete cDNA synthesis. The cDNA was stored at -20°C for later use.

[0037] Two pairs ...

Embodiment 3

[0040] Example 3 Construction of OsaUXS1 expression vector

[0041] According to the principle of homologous recombination of In-Fusion (Clontech Company), with the correctly sequenced plasmid pEASY-OsaUXS1 as a template, FETduetUXS1 (5'-gccaggatccgaattcgatggcgaaggagagctcc-3', SEQ ID NO.8) and RETduetUXS1 (5'-atgcggccgcaagcttttaactgctctgtttgctgg-3' , SEQ ID NO.9) as primers, PCR was carried out through the following procedures and systems, 50 μL system contained 5 μL of 10×PCR buffer, 5 μL of 2mM dNTPs, 25mM MgSO 4 3 μL, 1.5 μL each of 10 μM primers FETduetUXS1 and RETduetUXS1, 1 μL KOD-Plus-Neo, 1 μL plasmid, ddH 2 O make up. PCR program: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 30 s, renaturation at 63°C for 45 s, extension at 68°C for 120 s, a total of 30 cycles; extension at 68°C for 7 min, and incubation at 4°C. A 1 kb OsaUXS1 gene was amplified. The gene was connected to pET-Duet1 treated with EcoRI and HindIII double enzymes by the In-Fusion metho...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention provides a uridine-5'-diphosphate xylose synthase derived from Dieffenbachia tiger's eye, the amino acid sequence of which is shown in SEQ ID NO.1; The nucleotide sequence of the sugar synthase gene is shown in SEQ ID NO.3, as well as the vector and host cell containing the coding nucleotide sequence. The present invention also provides uridine-5'-diphosphate xylose synthase or a cell containing uridine-5'-diphosphate xylose synthase in catalytic substrate uridine-5'-diphosphate glucuronic acid reaction Applications.

Description

technical field [0001] The invention relates to a uridine-5'-diphosphate xylose synthase derived from Dieffenbachia tiger eye, its coding gene and its catalytic substrate uridine-5'-diphosphate glucuronic acid is uridine-5' -Application in xylose diphosphate reaction, which belongs to the field of genetic engineering. Background technique [0002] Uridine-5'-diphosphate xylose synthase (UXS) is the key enzyme to generate uridine-5'-diphosphate xylose (UDP-xylose) in organisms. UXS can decarboxylate uridine-5'-diphosphoglucuronic acid (UDP-glucuronic acid) to form UDP-xylose ( figure 1 ). UDP-xylose is an important glycosyl donor in the biosynthesis of glycoproteins, polysaccharides, and various glycosyl-modified secondary metabolites, such as flavonoid glycosides, triterpenoid saponins, and steroidal saponins. It exists widely in In animals, plants, fungi, bacteria. Many glycosylated compounds have better water solubility than their aglycones, which can increase the drug...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60
CPCC12N9/88C12Y401/01035
Inventor 孔建强尹森程克棣王伟
Owner INST OF MATERIA MEDICA CHINESE ACAD OF MEDICAL SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com