Glucose derepression inducible promoter and terminator and their application
A promoter and glucose technology, applied in the field of genetic engineering, can solve the problem that the promoter cannot be used
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Embodiment 1
[0098] Embodiment 1: the extraction of Rhodosporidium toruloides (Rhodosporidium toruloides) CGMCC 2.1389 total RNA
[0099] Rhodosporidium toruloides (R. toruloides) CGMCC 2.1389 (purchased from China General Microbiological Culture Collection Center (CGMCC)) was inoculated into 10 mL YEPD liquid medium (glucose 20.0 g / L , yeast extract 10.0g / L, peptone 20.0g / L, pH 6.0), cultured on a shaker at 30°C for 24h, and then transferred the bacterial solution to 100mL YEPD liquid medium at a volume ratio of 1:50 , and cultured on a shaker at 30°C for 14h to reach the logarithmic growth phase. Centrifuge at 5000rpm for 4min at 4°C to collect the cells, quickly freeze the cells with liquid nitrogen, and grind to break the wall (Yang F, Tan HD, Zhou YJ, et al.Mol.Biotechnol.2010,47(2):144– 151.). Total RNA was extracted using the TakaRa RNAiso kit and following its standard procedures.
[0100] The RNA was subjected to 1.5% (mass / volume concentration) agarose gel electrophoresis, obs...
Embodiment 2
[0101] Embodiment 2: Rhodosporidium toruloides CGMCC 2.1389cDNA first strand synthesis and ADH2 degenerate PCR
[0102] Using the total RNA of Rhodosporidium toruloides CGMCC 2.1389 as a template, the first strand of cDNA was synthesized by reverse transcription. First, mix 1.0 μL total RNA (about 2 μg), 1.0 μL primer SMART IV: 5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3′ and 1.0 μL oligo dT-linker primer CDSⅢ / 3′: 5′-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T) 30N-1 N-3′, 2.0 μL of DEPC-treated water (diethyl pyrocarbonate-treated water, purchased from Dalian TakaRa Company), was added to the PCR tube and mixed evenly, kept at 72°C for 2 minutes, immediately placed on ice for 2 minutes, and 2.0 μL of 5×first-strand buffer (Clontech), 1.0 μL of DTT (20 mM), 1.0 μL of dNTP (10 mM), and 1.0 μL of powerscript reverse transcriptase (Clontech) were added to the system and mixed evenly. Extend the reaction at 42°C for 60 minutes, and end the reaction at 4°C, and store it at -20°C for later us...
Embodiment 3
[0104] Example 3: Amplification of Rhodosporidium toruloides CGMCC 2.1389RtADH2 CDS
[0105] Genomic DNA of Rhodosporidium toruloides CGMCC 2.1389 was extracted by glass bead wall breaking method (Chapter 13 of the third edition of the Molecular Biology Experiment Guide, written by Osper et al., translated by Yan Ziying et al., published by Science Press). The prepared genomic DNA was measured by Nanodrop ND-1000, and the OD was measured 260 / OD 280 =1.85, indicating that the quality of genomic DNA is very good. The concentration was 280ng / μL, totaling 500μL, and the genomic DNA samples were frozen at -20°C for later use.
[0106] According to the cDNA sequence of alcohol dehydrogenase (RtADH2) induced by glucose deblocking obtained in Example 2, a pair of gene-specific primers were designed, ADH2-p1:5'-atgagcaaccctcaaatccccaaggaaggc-3' and ADH2-p2:5'-ttagaagttcttgaggacgatgcggcc -3', using the genomic DNA of Rhodosporidium toruloides CGMCC 2.1389 as a template, PCR amplific...
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