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Gracilaria chouae uridine diphosphate (UDP)-glucose pyrophosphorylase (UGPase) gene

A phosphorylase gene, uridine diphosphate technology, applied in genetic engineering, plant genetic improvement, hydrolase and other directions, can solve problems such as unconfirmed functions and lack of clones

Active Publication Date: 2014-04-09
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the functional verification of the uridine diphosphate glucose pyrophosphorylase (UGPase) gene in plants is mainly by detecting its activity in catalyzing the reverse reaction UDP-glucose and pyrophosphate to form glucose-1-phosphate and UTP, but in red algae and brown algae Cloning of the uridine diphosphate glucose pyrophosphorylase (UGPase) gene is scarce and its function remains unidentified

Method used

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  • Gracilaria chouae uridine diphosphate (UDP)-glucose pyrophosphorylase (UGPase) gene
  • Gracilaria chouae uridine diphosphate (UDP)-glucose pyrophosphorylase (UGPase) gene
  • Gracilaria chouae uridine diphosphate (UDP)-glucose pyrophosphorylase (UGPase) gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Cloning and analysis of the full-length coding region of the gene

[0026] Gracilaria crispus was collected from Shantou City, Guangdong Province in August 2011. The Trizol method was used to extract the total RNA from the sporophyte of Gracilaria crispatus, using PrimeScript II1 from TAKARA st The Strand cDNA Synthesis kit uses the first-strand cDNA reverse-transcribed from the total RNA of Gracilaria crispus sporophyte as a template, and uses Touchdown PCR technology to amplify the full-length CDS sequence of the GcUGP gene of Gracilaria crispus. The amplification primers include 2 sets ( 5'-TAGAATTCATGAATCGCGACTCCAGCTC-3' and 5'-TGCGGCCGCTTAATGCGG AATCAC-3'; 5'-TAGAATTCATGAATCGCGACTCCAGCTCG-3' and 5'-AGCGGCCGCATGCGGAATCACATG-3'). The PCR amplification program is: 94℃3min; 94℃30s, 60℃30s, 72℃2min, 15 cycles, each cycle reduces annealing temperature by 1℃; 94℃30s, 45℃30s, 72℃2min, 20 cycles ;72℃10min. After the PCR product is detected by 1% agarose gel ele...

Embodiment 2

[0027] Example 2: Preparation and analysis of GcUGP encoded protein

[0028] The GcUGP PCR product of Gracilaria fragile was detected by 1% agarose gel electrophoresis, and the target band was cut under UV light, and recovered by agarose gel. The recovered product GcUGP and pET32a plasmid were digested with BamHI and NotI at 37℃. After 3-4 hours of metal bath, use 1% agarose gel electrophoresis to detect, and use agarose gel recovery kit to recover. The target fragment GcUGP and plasmid pET32a were ligated, overnight at 16°C, and the constructed recombinant plasmid was named pET32a-GcUGP.

[0029] Transform the recombinant plasmid into E. coli expression strain BL21, pick BL21 positive clones, shake the bacteria and save the strain. PCR detects recombinants. The PCR products were detected by 1% agarose gel electrophoresis, and imaged by an automatic gel image analyzer. Pick the clones with the correct insert bands by electrophoresis for sequencing, check for mutations, and wheth...

Embodiment 3

[0031] Example 3: Functional verification of GcUGP encoded protein

[0032] UGP enzyme activity determination: The reaction system is as follows: 100mM 1×PBS, 0.85mM UDPG, 0.5mM PPi, 5mM Mgcl2, 0.3mM NADP, 5unit PGM, 5unit GDH and appropriate amount of recombinant GcUGP protein prepared in Example 2. The total reaction system is 1mL , Add substrate M-6-P to start the reaction. After mixing the above-mentioned substrate-removing system, incubate for 2 min at the corresponding temperature and start the reaction. Use the corresponding buffer as a blank control to measure the change in absorbance at 340nm for 0 min, 6 min and 12 min. Each reaction is set 4 parallel samples. After testing, the enzyme activity was 487.16U / g, the Km for UDPG was 1.67umol, the optimum reaction temperature was 40℃, and the optimum pH was 8.0. The enzyme is a high-temperature enzyme, basic protein; Mg 2+ , Ca 2+ , Mn 2+ And Zn 2+ Can promote enzyme activity, Pb 2+ , Cu 2+ Inhibit its activity.

[0033] At...

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Abstract

The invention relates to the field of genetic engineering technology, and particularly relates to a gracilaria chouae uridine diphosphate (UDP)-glucose pyrophosphorylase (UGPase) gene. The nucleotide sequence of the gene and the amino acid sequence of the coding protein are SEQ ID No.1 and SEQ ID No.2 respectively. According to the invention, the gene sequence is cloned through the gene cloning technology, and a prokaryotic expression vector is established; and enzyme activity detection on recombinant protein proves that the gene has the function of catalyzing UDP-glucose and pyrophosphoric acid to form glucose-1-phosphoric acid and UTP and belongs to a key enzyme coding gene of the synthesis path of agar-agar, starch, cellulose, trehalose, sucrose and the like. The gene has an important application value in increasing the content of economic components including algae agar-agar, starch, cellulose, trehalose, sucrose and the like.

Description

Technical field [0001] The invention relates to a uridine diphosphate glucose pyrophosphorylase gene of Gracilaria fragile. In particular, it relates to the nucleotide sequence of the uridine diphosphate glucose pyrophosphorylase gene of Gracilaria chouae, its coding protein, and its ability to synthesize starch, cellulose, trehalose, sucrose, etc. and are important for improvement Application in economic composition and stress resistance traits. Background technique [0002] Cloning products to synthesize related genes and verify their functions, reveal the relationship between genes and products, and assist in variety improvement has become one of the effective ways to increase the content of economic components in the field of international agricultural breeding; at the same time, the use of genetic engineering technology to produce active substances and economic The product has become the core content of the development of modern biotechnology industry. [0003] Uridine dipho...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/14C12P19/04
Inventor 刘涛池姗
Owner OCEAN UNIV OF CHINA
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