RNA polymerase III type promoter for rhodosporidium toruloides and application of RNA polymerase III type promoter
A technology of RNA polymerase and promoter, applied in the field of genetic engineering
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0067] Embodiment 1: the extraction of Rhodosporidium toruloides (Rhodosporidium toruloides) CGMCC 2.1389 genomic DNA
[0068] Rhodosporidium toruloides (R.toruloides) CGMCC 2.1389 (purchased from China General Microbiological Culture Collection Center (China General Microbiological Culture Collection Center, CGMCC)) was inoculated into 10mL YEPD liquid medium (glucose 20.0g / 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 24h to reach the logarithmic growth phase. Genomic DNA of Rhodosporidium toruloides was extracted according to the method in the literature (Lin XP, et al. FEMS Yeast Res 2014, 14:547–555.). The RNA was subjected to 1.5% (mass / volume concentration, 1.5g / 100ml) agarose gel electrophoresis, observed and identified using a fluorescence-ultraviolet analyzer, and a clear band was visibl...
Embodiment 2
[0069] Embodiment 2: Rhodosporidium toruloides CGMCC 2.1389 suspected promoter sequence amplification
[0070] Using Rhodosporidium toruloides CGMCC 2.1389 genomic DNA as a template, six suspected promoter sequences were amplified, named a, b, c, d, e, f, respectively. First, perform PCR amplification of fragment a: 5×PCR buffer 10.0μL, dNTPs (10mM) 1.0μL, upstream primer (a-F: 5'-TGGAGTTCGACGTTTCTCTCGC-3'50mmol / L) 1.0μL, downstream primer (a-R: 5 '- TGTGACTGATCTGGTGTTGTT-3' 50mmol / L) 1.0 μL, PrimeSTAR DNA polymerase (Dalian TakaRa) 0.5 μL, 1.0 μL genomic DNA as template, ddH 2 Add O to 50 μL, incubate at 94°C for 3 min, then incubate at 98°C for 10 s, 62°C for 10 s, 72°C for 1 min, 35 cycles, 72°C for 10 min, and end the reaction at 4°C. Fragments b, c, d, e, and f are amplified in the same way as fragment a, and the primers used are b-F: 5'-GGCGGGATGACCCAGCGCTTTCA-3' / b-R: 5'-CAAGACCGAAGTCGCTGGAAG-3', c-F: 5'- CCAGACGGACCTTGAGAACCC-3' / c-R: 5'-CCTCGCAAGCGGCCGACGCAGCC-3', d-F...
Embodiment 3
[0071] Embodiment 3: the amplification of Rhodosporidium toruloides CGMCC 2.1389 non-coding RNA nucleotide sequence
[0072] Using Genomic DNA of Rhodosporidium toruloides CGMCC 2.1389 as a template, two DNA sequences of miRNA and small nuclear RNA with different lengths were amplified, named A (59bp) and B (267bp), respectively. Since the siRNA fragment is small (35bp), it is directly synthesized by primers and named as D. First, perform PCR amplification of fragment A: 5×PCR buffer 10.0μL, dNTPs (10mM) 1.0μL, upstream primer (A-F: 5'-AAGCGCAACTACATCCTCG-3' 50mmol / L) 1.0μL, downstream primer (A-R: 5 '-CTCGTAGTCGATGATGCCGT-3' 50mmol / L) 1.0μL, PrimeSTAR DNA polymerase (Dalian TakaRa) 0.5μL, 1.0μL genomic DNA as a template, ddH2O was added to 50μL, incubated at 94°C for 3min, then at 98°C for 10s, 62°C for 10s, 72°C for 1min, 35 cycles, 72°C for 10min, 4°C to end the reaction. The amplification method of fragment B is the same as that of fragment A, and the primers used are re...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More