cDNA of freesiarefractaklatt UDP-dextrose flavone-3-o-glucosyltransferase
A glucose-based, glucose-based technology, applied in genetic engineering, plant genetic improvement, biochemical equipment and methods, etc., can solve problems such as low similarity of protein primary structure sequences
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Embodiment 1
[0025] Example 1 Cloning of the full-length FhGT1 gene
[0026] 1. Obtaining the probe: According to the Arabidopsis sequence, design specific primers, namely the upstream primer of 5'-CGA AGA CGG TGA AGA GGAC-3' (SEQ ID NO.5) and 5'-GGT GAC TGA AAC AAA The downstream primer of GAG CC-3' (SEQ ID NO. 6). Then the total RNA of Arabidopsis thaliana was extracted with the Trizol kit, and the first strand of cDNA was generated by reverse transcription, which was used as a template for RT-PCR, and the PCR product obtained was the probe for the screening library.
[0027] 2. Obtain the 5'sequence of the FhGT1 gene: Use the ECL luminescence kit and the above probes to screen the cDNA library of the petals of the vanilla safflower, pick the positive colonies obtained by exposure for sequencing, and the sequence is shown in SEQ ID NO.1 Show.
[0028] 3. Obtaining the 3'sequence of FhGT1 gene: According to the results of the above sequencing, design a specific upstream primer, namely 5'-AATCC...
Embodiment 2
[0030] Example 2 Expression of FhGT1 gene in E. coli cells
[0031] In order to express the FhGT1 protein, the cloned full-length FhGT1 gene was used as a template, and primers with EcoRI and HindIII restriction sites were used for PCR amplification of the FhGT1 gene open reading frame. The upstream primer: 5'-CTC GAA TTCCAG CAA GCA ATG GGA TCG-3' (SEQ ID NO. 11); the downstream primer 5'-CGG AAG CTT CAG ACT TCA GTCATA TTC CGA-3' (SEQ ID NO. 12).
[0032] The DNA fragments amplified above were digested with EcoR I and Hind III restriction enzymes, and cloned into plasmid pET-28(a+). The recombinant plasmid thus constructed was named pET-28a-FhGT1. After using restriction enzyme digestion and PCR identification, the results are as follows figure 2 Shown. Then, it was introduced into E. coli BL21 (DE3) cells, the transformant was cultured to express FhGT1 protein, the obtained inclusion bodies were lysed with 6M urea, and then slowly dialyzed to obtain recombinant FhGT1 protein.
Embodiment 3
[0033] Example 3 In vitro enzyme activity detection of FhGT1 protein
[0034] In vitro enzyme activity reaction system: (250μl)
[0035] 0.1M potassium phosphate buffer (pH7.0): 100μl
[0036] 10mM DTT: 25μl
[0037] 10mM cyanidin: 10μl
[0038] 10mM UDP-Glu: 25μl
[0039] 10% glycerol: 25μl
[0040] UFGT protein: 65μl
[0041] Reaction conditions: 30°C, 30 minutes, centrifuge at 14,000 rpm for 2 minutes after the reaction, the resulting supernatant was filtered with 0.22 μm, and then subjected to HPLC detection.
[0042] HPLC detection: detection using C18 column (specification: 250×4.6mm); detection conditions: 35°C, 270nm; elution conditions: the first 20 minutes linear elution: solution A (10% acetic acid + 0.1% phosphoric acid): 100-60% , Solution B (50% acetonitrile): 0-40%; isocratic elution after 20 minutes: solution A: 60%, solution B: 40%, the test results are as follows image 3 Shown. The test results showed that the in vitro enzyme biopsy experiment showed that anthocyanins w...
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