Galactokinase promoter and terminator, and application of promoter and terminator

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

Active Publication Date: 2017-01-11
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many researchers have isolated the galactokinase promoter of Saccharomyces cerevisiae or other ascomycetes, or other galactose-inducible promoters for their own genetic engineering operations (Liu Weifeng, Gao Dong, Wang Zunong. Bacteria System, 1998 ,17(3),256-261.; CN201410143885.0; CN201180008591.1; Schultz LD, Hofmann KJ, Mylin LM, et al.Gene.1987,61(2):123-133.; Choi ES1, Sohn JH , Rhee SK.ApplMicrobiol Biotechnol.1994,42(4):587-594.; Li J, Wang S, VanDusen WJ, et al.Biotechnol Bioeng.2000,70(2):187-196.), but no This type of promoter is used for gene expression, genetic engineering manipulation and genetic engineering of strain improvement in Rhodosoporidium, Sporidiobolus, Sporobolomyces and Rhodotorula operational reporting

Method used

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  • Galactokinase promoter and terminator, and application of promoter and terminator
  • Galactokinase promoter and terminator, and application of promoter and terminator
  • Galactokinase promoter and terminator, and application of promoter and terminator

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Embodiment 1: the extraction of Rhodosporidium toruloides (Rhodosporidium toruloides) CGMCC 2.1389 total RNA

[0100] 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, then transferred the bacterial solution to 100mL YEPD liquid medium at a volume ratio of 1:50, and Cultivate in a shaker at 30°C for 14 hours 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, etal.Mol.Biotechnol.2010,47(2):144–151 .). Total RNA was extracted using the TakaRa RNAiso kit and following its standard procedures.

[0101] The RNA was subjected to 1.5% (mass / volume concentration) agarose gel electrophoresis, obse...

Embodiment 2

[0102] Embodiment 2: Rhodosporidium toruloides CGMCC 2.1389cDNA first strand synthesis and GAL1 degenerate PCR

[0103] 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. Extend the reaction at 42°C for 60 minutes, and end the reaction at 4°C, and store it at -20°C for later use.

...

Embodiment 3

[0105] Example 3: Amplification of Rhodosporidium toruloides CGMCC 2.1389RtGAL1 CDS

[0106] 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 NanodropND-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.

[0107] According to the galactokinase (RtGal1) cDNA sequence obtained in Example 2, a pair of gene-specific primers were designed, GAL1-p1: 5'-atgccctcgctcgagacgtcgccgctc-3' and GAL1-p2: 5'-tcaatccgagttttggaagagcagtgcgcc-3', in circles Genomic DNA of Rhodosporidium CGMCC 2.1389 was used as a template, and PCR amplification was performed according to conve...

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Abstract

A promoter and a terminator which can be widely applied to gene expression, genetic engineering operation and strain improvement of Rhodosoporidium, Sporidiobolus, Sporobolomyces and Rhodotorula in red yeast through amplifying the upstream and downstream sequences of Rhodosporidium toruloides galactokinase genome DNA and carrying out biological information analysis and functional verification, the nucleotide sequence of the promoter is represented by SEQ ID NO:1, and the nucleotide sequence of the terminator is represented by SEQ ID NO:2. The invention also relates to DNA expression box or a recombinant vector containing above elements, a method using the correlated elements to construct Rhodosoporidium, Sporidiobolus, Sporobolomyces and Rhodotorula gene engineering strains, and corresponding strains.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a promoter terminator of Rhodosporidium toruloides and its application, including a transformation method necessary for the construction of genetic engineering strains and the like. Background technique [0002] Microorganisms are one of the most widely distributed species in nature. They have excellent biosynthetic ability and can synthesize almost all organic chemicals on earth. Compared with multicellular organisms, although the metabolic pathways of microorganisms are relatively simple, the production of their compounds is efficient and fast, with the characteristics of mild reaction conditions, strong controllability, and easy large-scale production, which can be used as an excellent cell factory. [0003] Some microorganisms in nature can store more than 20% of the dry cell weight of oil in the cell under certain conditions (such as nitrogen source d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/81C12N15/70C12N1/19C12N1/21
Inventor 赵宗保张素芳王雅南马斯佳
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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