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Na<+>/Pi cotransporter promoter and terminator as well as application thereof

A co-transporter and promoter technology, applied in the field of promoter terminators

Active Publication Date: 2016-11-23
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 some people have isolated the Na of Saccharomyces cerevisiae + / Pi cotransporter promoter, and other phosphate starvation-inducible promoters for their own genetic engineering manipulations (Hiraoka E, Murai M, Yano J, et al. Journal of Fermentation and Bioengineering.77(4):376–381 .; Lee KM, DaSilva NA.Yeast.2005,22(6):431–440.), but no promoters of this kind are used in Rhodosoporidium, Sporidiobolus, Reports of Gene Expression, Genetic Engineering Manipulation and Genetic Engineering Manipulation for Strain Improvement in Sporobolomyces and Rhodotorula

Method used

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  • Na&lt;+&gt;/Pi cotransporter promoter and terminator as well as application thereof
  • Na&lt;+&gt;/Pi cotransporter promoter and terminator as well as application thereof
  • Na&lt;+&gt;/Pi cotransporter promoter and terminator as well as application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Embodiment 1: the extraction of Rhodosporidium toruloides (Rhodosporidium toruloides) CGMCC 2.1389 total RNA

[0097] Rhodosporidium toruloides (R.toruloides) CGMCC 2.1389 (purchased from 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, then transferred the bacterial solution to 100mL YEPD liquid medium at a volume ratio of 1:50, and Incubate 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, et al.Mol.Biotechnol.2010,47(2):144– 151.). Total RNA was extracted using the TakaRa RNAiso kit and following its standard procedures.

[0098] The RNA was subjected to 1.5% (mass / volume concentration) agarose gel electrophoresis, observe...

Embodiment 2

[0099] Embodiment 2: Rhodosporidium toruloides CGMCC 2.1389cDNA first strand synthesis and pho89 degenerate PCR

[0100] Using the total RNA of Rhodosporidium toruloides NRRL Y-11557 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 5×first-strand buffer (Clontech), 1.0 μL DTT (20 mM), 1.0 μL dNTP (10 mM); 1.0 μL 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.

[0101] Design a...

Embodiment 3

[0102] Example 3: Amplification of Rhodosporidium toruloides CGMCC 2.1389 Genomic DNA

[0103] 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 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.

[0104] According to the Na obtained in Example 2 + / Pi cotransporter (RtPHO89) cDNA sequence, designed a pair of gene-specific primers, PHO89-p1: 5'-atgcctatgcaccaatacgattacctc-3' and PHO89-p2: 5'-ctactgcgaggggaagtgaggcgcgttgag-3', to Rhodosporidium toruloides CGMCC2 Genomic DNA of .1389 was used as a template, PCR amplification was performed according t...

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Abstract

The present invention amplifies Rhodosporidium toruloides Na + / Pi co-transporter genomic DNA upstream and downstream sequences, biological information analysis and functional verification, obtained can be widely used in Rhodosoporidium (Rhodosoporidium), lock throwing yeast (Sporidiobolus), throwing yeast ( Sporobolomyces) and Rhodotorula (Rhodotorula) gene expression, genetic engineering operations and strain improvement promoter and terminator, the nucleotide sequences of which are SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The present invention also relates to a DNA expression cassette or a recombinant vector containing these elements, and a method for constructing Rhodosporidium, Saccharomyces and Rhodotorula genetically engineered strains using related elements and corresponding bacterial 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): C12N15/113C12N15/11C12N15/63C12N1/21C12N1/19C12N15/81C12R1/645C12R1/19
Inventor 赵宗保王雅南张素芳马斯佳
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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