Gracilaria chouae uridine diphosphate (UDP)-glucose pyrophosphorylase (UGPase) gene
A phosphorylase gene and uridine diphosphate technology, applied in genetic engineering, plant genetic improvement, hydrolytic enzymes, etc., can solve problems such as lack of clones and unconfirmed functions
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Embodiment 1
[0025] Example 1: Cloning and analysis of the full-length coding region of the gene
[0026] Gracilaria crispa was collected from Shantou City, Guangdong Province in August 2011. The total RNA of Gracilaria brittle sporophyte was extracted by Trizol method, and PrimeScript II1 from TAKARA company was used st The Strand cDNA Synthesis kit uses the first-strand cDNA reverse-transcribed from the total RNA of Gracilaria brittle sporozoites as a template, and uses Touchdown PCR technology to amplify the full-length CDS sequence of the GcUGP gene of Gracilaria brittle, and 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°C for 3min; 94°C for 30s, 60°C for 30s, 72°C for 2min, 15 cycles, and the annealing temperature is lowered by 1°C in each cycle; 94°C for 30s, 45°C for 30s, 72°C for 2min, 20 cycles ; 72°C ...
Embodiment 2
[0027] Example 2: Preparation and analysis of GcUGP encoded protein
[0028] Gracilaria brittle GcUGP PCR product was detected by 1% agarose gel electrophoresis, and the target band was excised under ultraviolet light, recovered from the agarose gel, and the recovered product GcUGP and pET32a plasmid were subjected to BamHI and NotI double enzyme digestion, at 37°C After 3-4 hours in the metal bath, it was detected by 1% agarose gel electrophoresis and recovered using an agarose gel recovery kit. The target fragment GcUGP was ligated with the plasmid pET32a, overnight at 16°C, and the constructed recombinant plasmid was named pET32a-GcUGP.
[0029] 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 electr...
Embodiment 3
[0031] Example 3: Functional verification of GcUGP encoded protein
[0032] 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 GcUGP protein prepared in Example 2. The total reaction system is 1mL , the reaction was initiated by adding the substrate M-6-P. 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 487.16U / g, the Km to UDPG is 1.67umol, the optimum reaction temperature is 40°C, and the optimum pH is 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] ...
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