Uridine-5'-diphosphate apiose/xylose synthetic enzyme from ornithogalum caudatum, nucleotide sequence and application thereof

A diphosphate celery, synthesizing enzyme technology, applied in the direction of application, carbon-carbon lyase, enzyme, etc.

Active Publication Date: 2016-12-07
INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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And there is no report of isolating the prote...

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  • Uridine-5'-diphosphate apiose/xylose synthetic enzyme from ornithogalum caudatum, nucleotide sequence and application thereof
  • Uridine-5'-diphosphate apiose/xylose synthetic enzyme from ornithogalum caudatum, nucleotide sequence and application thereof
  • Uridine-5'-diphosphate apiose/xylose synthetic enzyme from ornithogalum caudatum, nucleotide sequence and application thereof

Examples

Experimental program
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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 strand was synthesized by adding buffer, dNTPs, RNase H and DNApolymerase I , after purification with QiaQuick PCR kit and elution with EB buffer, end repair, poly(A) was added and sequencing adapters were connected, and then agarose gel electrophoresis was used for fragment size selection, and finally PCR amplification was established. 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 raw reads. Perform data filtering on rawreads, remove reads with adapters, duplicates, and low-quality sequencing, and obtain clean rea...

Embodiment 2

[0035] Example 2 OsaUAXS1 gene cloning

[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 o...

Embodiment 3

[0040] Embodiment 3 Construction of OsaUXS1 expression vector

[0041] According to the principle of homologous recombination of In-Fusion (Clontech Company), using the correctly sequenced plasmid pEASY-OsaUXS1 as a template, FETduetUAXS1 (5'-gccaggatccgaattcgatgtcgtcgagagtggatct-3', SEQ ID NO.8) and RETduetUAXS1 (5'-atgcggccgcaagcttctagccagatgcaataggct-3 ', SEQ ID NO.9) as primers, carry out PCR through the following procedures and systems, 50 μL system contains 5 μL of 10×PCR buffer, 5 μL of 2mM dNTPs, 25mM MgSO 4 3 μL, 1.5 μL each of 10 μM primers FETduetUAXS1 and RETduetUAXS1, 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 1.2kb OsaUXS1 gene was amplified. The gene was connected to pET-Duet1 treated with EcoRI and HindIII double enzymes by the In-Fusion ...

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Abstract

The invention provides: an uridine-5'-diphosphate apiose/xylose synthetic enzyme from ornithogalum caudatum, of which an amino acid sequence is represented as the SEQ ID No.1; a nucleotide sequence, which is represented as the SEQ ID No.3, for encoding the gene of the uridine-5'-diphosphate apiose/xylose synthetic enzyme; a carrier and a host cell containing the encoding nucleotide sequence; and an application of the uridine-5'-diphosphate apiose/xylose synthetic enzyme or a cell containing same in a reaction of catalyzing a substrate uridine-5'-diphosphate glucuronic acid.

Description

technical field [0001] The invention relates to a uridine-5'-diphosphate apiose / xylose synthase derived from Dieffenbachia tiger's eye, its coding gene and its catalytic substrates uridine-5'-diphosphate glucuronic acid and nicotinamide The adenine dinucleotide is used in the reaction of uridine-5'-diphosphate xylose, uridine-5'-diphosphate celery sugar and reduced nicotinamide adenine dinucleotide, belonging to the field of genetic engineering. Background technique [0002] Uridine-5'-diphosphate apiose sugar / xylose synthase (UAXS) is the biological body to generate uridine-5'-diphosphate apiose sugar (UDP-apiose sugar), uridine-5'-diphosphate xylose ( UDP-xylose) is the key enzyme. UAXS can decarboxylate uridine-5'-diphosphoglucuronic acid (UDP-glucuronic acid) to form UDP-xylose, and catalyze UDP-glucuronic acid to undergo decarboxylation and carbon skeleton rearrangement to form UDP-apiose ( figure 1 ). UDP-xylose and UDP-apiose are two important sugars in the biosyn...

Claims

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Application Information

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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 AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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