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Orotidine-5'-phosphate decarboxylase promoter (pRtura3), application thereof, construct thereof and vector thereof

A technology of phosphate decarboxylase and orotic acid, applied in the field of genetic engineering, can solve problems such as difficult-to-target bacterial strain transformation

Active Publication Date: 2013-06-26
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

With Rhodosporidium toruloides as the host bacteria, whether it is genetic engineering operation or metabolic engineering strain improvement, it is restricted by the genetic operating system, and it is difficult to carry out targeted strain transformation

Method used

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  • Orotidine-5'-phosphate decarboxylase promoter (pRtura3), application thereof, construct thereof and vector thereof
  • Orotidine-5'-phosphate decarboxylase promoter (pRtura3), application thereof, construct thereof and vector thereof
  • Orotidine-5'-phosphate decarboxylase promoter (pRtura3), application thereof, construct thereof and vector thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: Preparation of Rhodosporidium toruloides genomic DNA

[0040] Inoculate fresh Rhodosporidium toruloides ATCC 10788 (purchased from the American Standard Biological Collection Center, ATCC) into 50ml YEPD liquid medium from a slant, culture it on a shaker at 30°C for 24 hours, and then inoculate it at a volume of 1:50 Ratio Transfer the bacterial solution to 100ml YEPD liquid medium respectively, and culture it on a shaker at 30°C for 12h to reach the logarithmic growth phase. Centrifuge at 5000rpm for 4min at 4°C to collect the cells. The genomic DNA of ATCC 10788 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 of genomi...

Embodiment 2

[0041] Embodiment 2: Chromosomal walking obtains Rtura3 gene 5' flank sequence (promoter)

[0042] This example was completed using Genome Walking Kit (purchased from TaKaRa).

[0043] According to the genomic DNA sequence of the reported Rhodosporidium toruloides orotidine-5'-phosphate decarboxylase coding gene (Rtura3) (Chinese patent, patent application number: 200710158415.1; Yang F, Zhang S, Tang W, Zhao Z. Identification of theorotidine-5'-monophosphate decarboxylase gene of the oleaginous yeast Rhodosporidium toruloides Yeast 2008, 25(9): 623-630), design 3 SpecificPrimers (gene-specific primers) respectively ura3-SP1: 5'-TCCCGGTCAAAGTCCTCGACGATGTC -3', ura3-SP2: 5'-CTTGATGCAGCAGCAGTACGGGCC-3' and ura3-SP3: 5'-GGCGTAGGTGCGGGTCGTGATGGAC-3' were used as downstream primers, and the following operations were performed according to the instructions of the GenomeWalking Kit (purchased from TaKaRa).

[0044] 1.1 st PCR reaction

[0045] The first round of amplification was ...

Embodiment 3

[0082] Embodiment 3: Chromosomal walking obtains Rtura3 gene 3' flank sequence (terminator)

[0083] This example is also completed using Genome Walking Kit (purchased from TaKaRa).

[0084] Genomic DNA sequence of the reported gene encoding Rhodosporidium toruloide orotidine-5′-phosphate decarboxylase (Rtura3) (Chinese patent, patent application number: 200710158415.1; Yang F, Zhang S, Tang W, Zhao Z. Identification of the orotidine-5'-monophosphatedecarboxylase gene of the oleaginous yeast Rhodosporidium toruloides Yeast2008, 25(9):623-630), design 3 Specific Primers (gene-specific primers) respectively ura3-SP11: 5'-GGAGGGTGGACGAGCGGGAAGAGAC- 3', ura3-SP22: 5'-GTCATCATGACGC CCGGCGTCGGACT-3' and ura3-SP33: 5'-GCGTACGAGGACAGGCTG AGGCAGT-3', as upstream primers, perform 3' wing chromosome walking according to the instructions of GenomeWalking Kit (purchased from TaKaRa) The operation is the same as in Example 2, except that the SpecificPrimer is replaced by ura3-SP1, ura3-SP2...

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Abstract

By amplifying an upstream sequence and a downstream sequence of genome DNA of a pRtura3 of Rhodosporidium tondoides, the biological information analysis and function verification is carried out to obtain a promoter and a terminator which can effectively express a target gene in Rhodosporidium tondoides to be used in genetic engineering operation and strain improvement of Rhodosporidium tondoides.The invention also relates to a construct and a vector of DNA containing elements.

Description

technical field [0001] This patent belongs to the technical field of genetic engineering, and specifically relates to Rhodosporidium toruloides promoters, terminators and their uses, including transformation methods necessary for the construction of genetic engineering strains. 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. The species diversity and genetic diversity of microorganisms determine their metabolic diversity. Compared with multicellular organisms, although the metabolic pathways of microorganisms are relatively simple, the production of their compounds is efficient and fast, and is closely related to human daily production and life. [0003] As a natural production strain of a certain chemical or an environmental treatment application strain, its specific production performance is often not optimal. How to op...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12R1/645
Inventor 赵宗保杨帆张素芳
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI