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Rhodosporidium toruloides RNA promoter and application thereof

A technology of Rhodosporidium toruloides and promoters, applied in DNA/RNA fragments, recombinant DNA technology, introduction of foreign genetic material using vectors, etc., to achieve the effect of targeted gene knockout

Inactive Publication Date: 2020-12-29
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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Problems solved by technology

To date, the CRISPR / Cas9 gene editing system has been successfully applied to most model and quasi-model yeasts, including Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Kluyvis lactis, S. pombe, Candida albicans Yeast, Cryptococcus neoformans, etc. (Stovicek V, Holkenbrink C, Borodina I, FEMS yeast research, 2017, 17:1-16), but not reported in Rhodosporidium toruloides

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  • Rhodosporidium toruloides RNA promoter and application thereof
  • Rhodosporidium toruloides RNA promoter and application thereof
  • Rhodosporidium toruloides RNA promoter and application thereof

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Embodiment

[0041] 1. Materials and methods

[0042] 1.1 Bacteria and culture conditions

[0043] Rhodosporidium toruloides strain NP11 (purchased from Wuhan Microbial Culture Collection Center) was used as the material. Routine culture uses YPD liquid medium (glucose 20g / L, peptone 20g / L, yeast extract 10g / L), and the culture conditions are 28°C, 250r / min.

[0044] 1.2 Prediction of gRNA promoters

[0045] Using the DNA conserved sequences recognized by the RNA polymerase III transcription factor TFIIIC of Saccharomyces cerevisiae and Yarrowia lipolytica as a reference, using the Optimize for Somewhat similar sequences program in the NCBI Blastn package, obtained from the genome data of Rhodosporidium toruloides Homologous sequences of Rhodosporidium toruloides genome. From the genome sequence of Rhodosporidium toruloides in the database, the Rhodosporidium toruloides tRNA gene was obtained. The sequence is as follows:

[0046] Taking the promoter of the Yarrowia lipolytica U6 gene ...

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Abstract

The invention relates to the field of biotechnology, in particular to a Rhodosporidium toruloides RNA promoter and application thereof. The promoter is Rhodosporidium toruloides RNA polymerase III promoter; and it is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or a DNA nucleotide sequence which has 70% homology with the above sequences and has promoter activity. The promoter of the invention is applied to the CRISPR / cas9 gene editing system of Rhodotorula rotundifolia and to guide the expression of gRNA in the system. The gene editing efficiency can reach 80%, and efficient site-specific gene knockout can be achieved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an RNA promoter of Rhodosporidium toruloides and its application. Background technique [0002] Rhodosporidium toruloides is a typical oleaginous yeast, which can synthesize lipids with high cell content under the limitation of various nutrients. Under the limitation of nitrogen elements, the accumulation of lipids can reach 70% of the dry weight of the cells. Saccharomyces solani can utilize a variety of carbon sources, such as lignocellulose hydrolyzate, glycerol, organic acids, hexoses, and the like. Rhodosporidium toruloides can also synthesize carotenoids, including β-carotene, lutein, Torularhodin, etc. These pigments have various functions such as anti-oxidation and improving immunity. [0003] At the end of the 20th century, some scholars carried out genetic engineering research on Rhodosporidium toruloides, for example: chemical mutagenesis of the yeast to obtain auxotrophi...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/81C12N1/19C12R1/645
CPCC07K14/39C12N15/815
Inventor 王士安赵晨李福利
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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