A truncated uridine-5'-diphosphate xylose synthase, its nucleotide sequence and application

Abstract

The present invention provides a truncated uridine-5'-diphosphate xylose synthase derived from Dieffenbachia tiger's eye, the original amino acid sequence of which is shown in SEQ ID NO.1; it also provides a coded truncated uridine The nucleotide sequence of ‑5'‑diphosphate xylose synthase gene, as shown in SEQ ID NO.2, as well as the vector and host cell containing the coding nucleotide sequence. The present invention also provides truncated uridine-5'-diphosphate xylose synthase or cells containing truncated uridine-5'-diphosphate xylose synthase in catalytic substrate uridine-5'-diphosphate Application in phosphoglucuronic acid reaction.

Description

technical field

[0001] The invention belongs to the field of genetic engineering. Involving the truncated uridine-5'-diphosphate xylose synthase in Dieffenbachia tiger's eye, its encoding gene and its catalytic substrate uridine-5'-diphosphate glucuronic acid to uridine-5'-diphosphate Application in xylose phosphate reaction. 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...

Examples

Embodiment 1

[0032] Example 1. Transcriptome Sequencing and Sequence Analysis

[0033] 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.

[0034] 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 clean reads. Use the short ...

Embodiment 2

[0036] Example 2 Cloning of OsaUXS3 gene

[0037] 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.

[0038] Two pairs...

Embodiment 3

[0041] Example 3 Construction of OsaUXS3 expression vector

[0042] The results of multiple sequence alignment showed that, compared with the protein sequence of cytoplasmic UXS, OsaUXS3 (SEQ ID NO.9) had about 100 more amino acid sequences at the nitrogen terminal ( image 3 ). With TMHMM( http: / / www.cbs.dtu.dk / services / TMHMM / ) predicted the transmembrane region of OsaUXS3 protein, the results showed that OsaUXS3 had an obvious transmembrane region, and OsaUXS3 protein was predicted to be a type II membrane protein ( Figure 4 ). Therefore, in order to express the protein as a soluble protein in the prokaryotic system, it is necessary to remove its transmembrane region when designing In-Fusion primers.

[0043] According to the principle of homologous recombination of In-Fusion (Clontech Company), using the correctly sequenced plasmid pEASY-OsaUXS3 as a template, FET28aUXS3 (5'-tcgcggatccgaattcatgcacccttacttctcttac-3', SEQ ID NO.10) and RET28aUXS3 (5'-gtgcggccgcaagctt...

technical field

[0001] The invention belongs to the field of genetic engineering. Involving the truncated uridine-5'-diphosphate xylose synthase in Dieffenbachia tiger's eye, its encoding gene and its catalytic substrate uridine-5'-diphosphate glucuronic acid to uridine-5'-diphosphate Application in xylose phosphate reaction. 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...

Examples

Embodiment 1

[0032] Example 1. Transcriptome Sequencing and Sequence Analysis

[0033] 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.

[0034] 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 clean reads. Use the short ...

Embodiment 2

[0036] Example 2 Cloning of OsaUXS3 gene

[0037] 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.

[0038] Two pairs...

Embodiment 3

[0041] Example 3 Construction of OsaUXS3 expression vector

[0042] The results of multiple sequence alignment showed that, compared with the protein sequence of cytoplasmic UXS, OsaUXS3 (SEQ ID NO.9) had about 100 more amino acid sequences at the nitrogen terminal ( image 3 ). With TMHMM( http: / / www.cbs.dtu.dk / services / TMHMM / ) predicted the transmembrane region of OsaUXS3 protein, the results showed that OsaUXS3 had an obvious transmembrane region, and OsaUXS3 protein was predicted to be a type II membrane protein ( Figure 4 ). Therefore, in order to express the protein as a soluble protein in the prokaryotic system, it is necessary to remove its transmembrane region when designing In-Fusion primers.

[0043] According to the principle of homologous recombination of In-Fusion (Clontech Company), using the correctly sequenced plasmid pEASY-OsaUXS3 as a template, FET28aUXS3 (5'-tcgcggatccgaattcatgcacccttacttctcttac-3', SEQ ID NO.10) and RET28aUXS3 (5'-gtgcggccgcaagctt...

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