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A kind of rhodosporidium toruloides rna polymerase type III promoter and application thereof

A technology of promoter and polymerase, applied in the field of genetic engineering

Active Publication Date: 2022-06-07
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Although the RNA polymerase III promoter of Saccharomyces cerevisiae has been isolated to transcribe mature sgRNA, it guides the Cas9 protein to cut the target site, thereby generating gene editing (DiCarlo JE, etal. Nucleic Acids Research 2013, 41: 4336–4343.), but no such promoters have been used for gene expression in Rhodosoporidium, Sporidiobolus, Sporobolomyces and Rhodotorula, Reports of genetic engineering manipulations and genetic engineering manipulations for strain improvement

Method used

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  • A kind of rhodosporidium toruloides rna polymerase type III promoter and application thereof
  • A kind of rhodosporidium toruloides rna polymerase type III promoter and application thereof
  • A kind of rhodosporidium toruloides rna polymerase type III promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: Extraction of Rhodosporidium toruloides CGMCC 2.1389 genomic DNA

[0068] R. toruloides CGMCC 2.1389 (purchased from China General Microbiological Culture Collection Center, CGMCC) was inoculated into 10 mL of YEPD liquid medium (glucose 20.0 g / L) from a slant. , yeast extract 10.0g / L, peptone 20.0g / L, pH 6.0), incubate at 30°C for 24h on a shaker, and then transfer the bacterial liquid to 100mL YEPD liquid medium at a volume ratio of 1:50. , cultured at 30°C in a shaker for 24h to reach the logarithmic growth phase. The extraction of Rhodosporidium toruloides genomic DNA was performed according to the method in the literature (Lin XP, et al. FEMS Yeast Res 2014, 14:547–555.). 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 seen. Use NanoDrop to measure the quality and concentration of genomic DNA, the measured concentration...

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 and named as a, b, c, d, e, and f respectively. First, perform PCR amplification of fragment a: 10.0 μL of 5×PCR buffer, 1.0 μL of dNTPs (10 mM), 1.0 μL of upstream primer (a-F: 5’-TGGAGTTCGACGTTCTCCTCGC-3’ 50 mmol / L), 1.0 μL of downstream primer (a-R: 5 '- TGTGACTGATCTGGTGTTGTT-3'50mmol / L) 1.0μL, PrimeSTAR DNA polymerase (Dalian TakaRa) 0.5μL, 1.0μL genomic DNA as template, ddH 2 O supplemented to 50 μL, incubated at 94 °C for 3 min, then incubated at 98 °C for 10 s, 62 °C for 10 s, 72 °C for 1 min, 35 cycles, 72 °C for 10 min, and 4 °C to complete the reaction. The amplification of the b, c, d, e, and f fragments is the same as the amplification of the a fragment, and the primers used are b-F: 5'-GGCGGGATGACCCAGCGCTTTCA-3' / b-R: 5'-CAAGACCGAAGTCGCT...

Embodiment 3

[0071] Example 3: Amplification of Rhodosporidium toruloides CGMCC 2.1389 non-coding RNA nucleotide sequence

[0072] Using the genome DNA of Rhodosporidium toruloides CGMCC 2.1389 as the template, two DNA sequences of miRNA and small nuclear RNA with different lengths were amplified, which were named A (59bp) and B (267bp). Since the siRNA fragment was small (35bp), it was directly synthesized by primers and named D. First, perform PCR amplification of fragment A: 5× PCR buffer 10.0 μL, dNTPs (10 mM) 1.0 μL, upstream primer (A-F: 5’-AAGCGCAACTACATCCTCG-3’ 50 mmol / L) 1.0 μL, downstream primer (A-R: 5 '-CTCGTAGTCGATGATGATGCCGT-3'50mmol / L) 1.0μL, PrimeSTAR DNA polymerase (Dalian TakaRa) 0.5μL, 1.0μL genomic DNA as template, ddH2O supplemented to 50μL, incubated at 94°C for 3min, then incubated at 98°C for 10s, 62 °C for 10 s, 72 °C for 1 min, 35 cycles, 72 °C for 10 min, and 4 °C to complete the reaction. The amplification of the B fragment is the same as the amplification of ...

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Abstract

In the present invention, by amplifying a segment of Rhodosporidium toruloides DNA sequence and performing biological function verification, the obtained genus Rhodosoporidium, Sporidiobolus, and Saccharomyces ( Sporobolomyces) and Rhodotorula (Rhodotorula), RNA polymerase type III promoters for non-coding RNA transcription, genetic engineering operations and strain improvement, the nucleotide sequence of which is SEQ ID NO:1. The present invention also relates to a method for constructing genetically engineered strains of the genus Rhodosporidium, Saccharomyces genus and Rhodotorula genus and the corresponding strains containing the DNA sequence expression cassette or recombinant vector, and using related elements.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to an RNA polymerase type III promoter of Rhodosporidium toruloides and its use, including transformation methods necessary for the construction of genetically engineered strains and the like. Background technique [0002] Microorganisms are one of the most widely distributed species in nature, and some of them can store more than 20% of their dry weight of lipids in their cells under certain conditions (such as lack of nitrogen and phosphorus sources), among which triglycerides are the main ones. , microorganisms with this phenotype are called oleaginous microorganisms, including bacteria, yeast, molds, algae, etc. (Ratledge C and Wynn JP. Adv Appl Microbiol 2002, 51:1–51.). The use of microorganisms to convert biomass resources to produce oil can be developed into a new technology that basically does not depend on arable land, can be continuously produced, reduce agric...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/70C12N15/81C12N1/21C12N1/19
CPCC12N9/1276C12N15/70C12N15/815C12Y207/07006Y02E50/10
Inventor 赵宗保焦翔张素芳
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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