Galactokinase promoter and terminator and their application

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

Active Publication Date: 2019-03-05
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 Saccharomyces cerevisiae or other ascomycete galactokinase promoters, 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, MylinLM, et al.Gene.1987,61(2):123-133.; Choi ES1, Sohn JH , Rhee SK.Appl MicrobiolBiotechnol.1994,42(4):587-594.; Li J, Wang S, VanDusen WJ, et al.BiotechnolBioeng.2000,70(2):187-196.), but did not see the Genetic engineering of gene expression, genetic engineering and strain improvement in Rhodosoporidium, Sporidiobolus, Sporobolomyces and Rhodotorula report

Method used

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  • Galactokinase promoter and terminator and their application
  • Galactokinase promoter and terminator and their application
  • Galactokinase promoter and terminator and their application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083]Embodiment 1: the extraction of Rhodosporidium toruloides (Rhodosporidium toruloides) CGMCC 2.1389 total RNA

[0084] 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.

[0085] The RNA was subjected to 1.5% (mass / volume concentration) agarose gel electrophoresis, obse...

Embodiment 2

[0086] Embodiment 2: Rhodosporidium toruloides CGMCC 2.1389 cDNA first strand synthesis and GAL1 degenerate PCR

[0087] 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

[0089] Example 3: Amplification of Rhodosporidium toruloides CGMCC 2.1389RtGAL1 CDS

[0090] 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.

[0091] 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'-tcaatccgagttctggaagaggagtgc-3', in circles Genomic DNA of Rhodosporidium CGMCC 2.1389 was used as a template, and PCR amplification was performed according to convent...

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Abstract

By amplifying the upstream and downstream sequences of genome DNA of galactokinase of Rhodosporidium toruloides, a promoter and a terminator are obtained. The promoter and terminator can be widely applied to gene expression, operation of genetic engineering, and strain improvement of Rhodosoporidium, Sporidiobolus, Sporobolomyces, and Rhodotorula. The nucleotide sequences of the promoter and the terminator are SEQ ID No.1 and SEQ ID No.2 respectively. The invention also relates to a DNA expression cassette or recombinant carrier comprising the elements, a method utilizing related elements to establish gene engineering strains of Rhodosoporidium, Sporidiobolus, and Rhodotorula, 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 Patents(China)
IPC IPC(8): C12N15/113C12N15/70C12N15/81C12N1/19C12N1/21
Inventor 张素芳赵宗保马斯佳王雅南
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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