Kelp uridine diphosphate (UDP)-glucose pyrophosphorylase (UGPase) gene

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

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 the same time, in the biosynthesis of important seaweed gel-agar, uridine diphosphate glucose pyrophosphorylase (UGPase) is considered to be the rate-limiting enzyme in the process of fucoside synthesis, controlling the important product UDP-D- The synthesis of glucose is of great value for increasing the agar content in red algae. At present, the functional verification of the uridine diphosphate glucose pyrophosphorylase (UGPase) gene in plants is mainly by detecting its catalytic reverse reaction UDP-glucose and pyrophosphate The activity of forming glucose-1-phosphate and UTP, but the cloning of uridine diphosphate glucose pyrophosphorylase (UGPase) gene in red algae and brown algae is very lacking, and its function has not yet been confirmed

Method used

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  • Kelp uridine diphosphate (UDP)-glucose pyrophosphorylase (UGPase) gene
  • Kelp uridine diphosphate (UDP)-glucose pyrophosphorylase (UGPase) gene
  • Kelp uridine diphosphate (UDP)-glucose pyrophosphorylase (UGPase) gene

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1: Cloning and analysis of the full-length coding region of the gene

[0027] The kelp was collected from Rongcheng City, Shandong Province in July 2011. Laminaria female gametophyte total RNA was extracted by Trizol method, using TAKARA PrimeScript II 1 stThe Strand cDNA Synthesis kit uses the first-strand cDNA reverse-transcribed from the total RNA of Laminaria gametophytes as a template, and uses Touchdown PCR technology to amplify the full-length CDS sequence of the Laminaria SjUGP gene. The amplification primers include 2 sets (5′-CAGCGGATCCATGTCTAGTGTCAGCATGAACGCGG- 3′ and 5′-ATTAGCGGCCGCCGCACCCTCAGCCGA-3′; 5′-CAGCGGATCCATGTCTAGTGTCAGCATGAACGCGG-3′ and 5′-ATTAGCGGCCGCCGCACCCTCAGCCGA-3′). The PCR amplification program was: 94°C for 3min; 94°C for 30s, 60°C for 30s, 72°C for 2min, 15 cycles, and the annealing temperature was lowered by 1°C in each cycle; 94°C for 30s, 45°C for 30s, 72°C for 2min, 20 cycles ; 72°C for 10 min. After the PCR products were de...

Embodiment 2

[0028] Example 2: Preparation and Analysis of SjUGP Encoded Protein

[0029] After the Kelp SjUGP PCR product was detected by 1% agarose gel electrophoresis, the target band was excised under ultraviolet light, recovered from the agarose gel, and the recovered product SjUGP and pET32a plasmid were digested with EcoRI and NotI, and placed in a metal bath at 37°C After 3-4 hours, it was detected by 1% agarose gel electrophoresis and recovered using an agarose gel recovery kit. The target fragment SjUGP was ligated with the plasmid pET32a, overnight at 16°C, and the constructed recombinant plasmid was named pET32a-SjUGP.

[0030] Transform the recombinant plasmid into Escherichia coli expression strain BL21, pick BL21 positive clones, shake the bacteria and save the strain. PCR detection of recombinants. PCR products were detected by 1% agarose gel electrophoresis and imaged by an automatic gel image analyzer. Pick and sequence the correct clones for electrophoresis detection ...

Embodiment 3

[0032] Example 3: Functional verification of SjUGP encoded protein

[0033] Determination of UGP enzyme activity: 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 an appropriate amount of recombinant SjUGP protein prepared in Example 2. The total reaction system is 1 mL, the substrate M-6-P was added to initiate the reaction. Mix the above substrate-removing system and incubate at the corresponding temperature for 2 minutes to initiate the reaction. Use the corresponding buffer as a blank control and measure the changes in the absorbance at 340 nm at 0 min, 6 min and 12 min. 4 parallel samples. After testing, the enzyme activity is 373.38U / g, the Km to UDPG is 4.33umol, the optimum reaction temperature is 37°C, and the optimum pH is 8.0. The enzyme is a high temperature enzyme, basic protein; Mg 2+ , Zn 2+ , Mn 2+ and Ca 2+ Can promote enzyme activity, Pb 2+ 、Cu 2+ inhibit its activity.

[0034] At...

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Abstract

The invention relates to the field of a genetic engineering technology, and particularly relates to a kelp 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 kelp uridine diphosphate glucose pyrophosphorylase gene. In particular, it relates to the nucleotide sequence of a kelp (Saccharina japonica) uridine diphosphate glucose pyrophosphorylase gene, its encoded protein, and its ability to synthesize starch, cellulose, trehalose, sucrose, etc., and to improve important economic components and applications in stress resistance traits. Background technique [0002] Kelp is the predominant species of marine plants (algae) cultivated worldwide. As a marine vegetable, kelp has high nutritional value. At the same time, it contains alginate, starch, cellulose and other economic components, which can be widely used as raw materials in food, medicine and hygiene, textile industry, scientific research and other aspects. [0003] Cloning products to synthesize related genes and verify their functions, revealing the relationship between genes and products, and assisting in the improvement of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/14C12P19/04
Inventor 刘涛池姗
Owner OCEAN UNIV OF CHINA
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