Chimeric gene with delta 6 fatty acid dehydrogenase function and its use
A chimeric gene and amino acid technology, applied to a chimeric gene with Δ6 fatty acid dehydrogenase function and its application field, can solve the problems of low GLA yield and the like
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Embodiment 1
[0035] Example 1. Construction of chimeric genes
[0036] 1. Preparation of black currant genomic DNA
[0037]Take the living black tea currant (Ribes nigrum L.) plant grown in Beijing Botanical Garden as the material, take about 100 mg of its young leaves, add steel balls (diameter 5mm) into a 7mL Ep tube, and freeze in liquid nitrogen for 20-30min , high-speed crushing on the vortex, repeat the operation 2-3 times until the material is completely crushed. Add 1-2mL of preheated CTAB extract solution (see "Refined Molecular Biology Experiment Guide" 2001, Science Press, translated by Yan Ziying and Wang Hailin). Mix well. 65°C water bath for 30min. Add an equal volume of chloroform, and gently extract for about 5 minutes. Centrifuge at 12000rpm room temperature for 10min. Take the supernatant, add 1 / 2 volume of isopropanol to mix well, and place at room temperature for 10 min to precipitate DNA. The precipitated DNA was picked out with a Tip, washed twice with 70% ethan...
Embodiment 2
[0083] Embodiment 2. Construction of chimeric gene yeast vector
[0084] From the pMD18-T vector containing the chimeric genes RnD6CA and RnD6CB, the HindIII and Xba I restriction sites were used to obtain the RnD6CA and RnD6CB genes after double-enzyme digestion, and they were directional cloned into the yeast expression vector pYES2.0 (purchased from Invitrogen Co., Ltd. ), obtained yeast expression plasmids pYRnD6CA and pYRnD6CB, transformed into Escherichia coli DH-5α for preservation. For its carrier map, see Figure 8 .
Embodiment 3
[0085] Example 3. Expression of chimeric genes in yeast
[0086] 1. Transformation of Yeast
[0087] Referring to the method described in Invitrogen's pYES2 Kit (Cat#V285-20), the yeast expression plasmids pYRnD6CA and pYRnD6CB of the above-mentioned chimeric genes (see Figure 8 ), using lithium acetate to mediate the transformation of Saccharomyces cerevisiae auxotrophic strain INVSc I (referring to the transformation method described in the pYES2 Kit of Invitrogen Company) (purchased from Invitrogen Company), and using the empty vector pYES2.0 plasmid as a control, to obtain expression plasmids containing each yeast cells.
[0088] 2. Induced expression in yeast transformed cells
[0089] Take the yeast single colony transformed with the yeast expression plasmid containing the chimeric gene, inoculate it in 50 ml of SC-U culture medium containing 2% raffinose (refer to the formula described in Invitrogen's pYES2 Kit), and cultivate it overnight at 250 rpm on a shaker at 2...
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