Glycosyltransferases of flavonoids from Dianthus cuneiformis and its coding genes and applications
A technology of glycosyltransferase and compound, which is applied in the fields of genetic engineering and enzyme engineering to achieve the effects of high catalytic efficiency, broad application prospects and high economic value
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Embodiment 1
[0053] Example 1. Cloning of the expression gene MemUGT1
[0054] 1.1 CTAB-PVP method to extract total RNA from C. wedgelobata
[0055] (1) Take the plant thallus of Dictyostelium wedges grown in the greenhouse for two months, wash it clean, freeze it with liquid nitrogen, and then place it in a mortar and grind until the material becomes powdery.
[0056] (2) Take an appropriate amount of plant powder into a 2 mL imported centrifuge tube pre-cooled in advance, add 800 μl of CTAB-PVP extract solution pre-heated at 65°C, and mix upside down.
[0057] The above-mentioned CTAB-PVP extraction buffer preparation method is as follows:
[0058] 100 mM Tris HCl (pH 8.0), 2% CTAB (w / v), 2% PVP (w / v), 25 mM EDTA, 2 M NaCl, mercaptoethanol was added to 0.2% after autoclaving; solution preparation was treated with DEPC ddH 2 O, spare after autoclaving.
[0059] (3) 65℃ water bath for 30min, invert and mix once every 6-10min.
[0060] (4) After cooling, add 600 μl of chloroform and mi...
Embodiment 2
[0110] Example 2. Analysis of gene protein expression and enzyme activity function
[0111] 2.1 Extraction of MemUGT1-pTOPO plasmid
[0112] Plasmids were extracted with a plasmid mini-extraction kit (TIANGEN):
[0113] (1) The existing strains MemUGT1-pTOPO-DH5α were divided into LB plates (containing 100 μg / mL Amp), 37 ° C, after 12 hours, single clones were grown, and single clones were picked in 4 mL of Amp-resistant medium, 37 ° C , 110rpm cultured for 10h.
[0114] (2) Centrifuge the bacterial solution at 12,000 rpm for 1 min at room temperature, discard the supernatant, collect the bacterial cells, and discard the supernatant as much as possible.
[0115] (3) Add 150 μL of solution P1 to the centrifuge tube with the bacterial cell precipitation left, and vortex to shake until the bacterial cell is completely suspended.
[0116] (4) Add 150 μL of solution P2 to the centrifuge tube, and gently turn up and down 6-8 times to fully lyse the cells.
[0117] (5) Add 350 μL...
Embodiment 3
[0185] Example 3. Biosynthesis of flavonol 3-O-glucoside by MemUGT1
[0186] 3.1 Production of compounds using Escherichia coli MemUGT1-pET32a-BL21(U1)
[0187] In order to study the effects of medium type, substrate concentration and in vivo feeding and incubation time on glycoside products, we carried out in vivo feeding experiments of recombinant strain U1 with substrates quercetin and kaempferol respectively. The specific experimental operations are as follows:
[0188] (1) Activate the strain in a 37°C constant temperature incubator, pick a single clone and inoculate it into 4mL LB liquid medium, TB liquid medium and M9 liquid medium (containing Amp 100μg / mL), and continue to cultivate in a 37°C incubator 7h;
[0189] (2) The target strain and the control strain were inoculated into 50 mL of resistant LB medium, TB medium and M9 medium according to the ratio of 1:100, and cultured to OD in a shaker at 37°C and 200 rpm. 600 =0.4-0.6, add IPTG to make the final concentrat...
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