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Promoter and terminator of 3-phosphoglyceraldehyde dehydrogenase and application thereof

A glyceraldehyde phosphate dehydrogenase and promoter technology, which is applied in the field of genetic engineering, can solve the problems such as the report of the promoter sequence of T. dermoides, restricting strain transformation, and the lack of promoter, and achieves the promotion of strain improvement and Effects of metabolic engineering research

Active Publication Date: 2016-07-20
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

But so far, no promoter sequence suitable for Trichosporium dermatitis has been reported, nor is there any endogenous promoter suitable for Lipoyces stariae, which restricts the targeted strain transformation
[0006] Although there have been people who have isolated the 3-phosphate glyceraldehyde dehydrogenase promoters of Rhodosporidium toruloides and Rhodotorula glutinosa for their own genetic engineering operations (FEMS Yeast Res. 2014, 14 (4): 547-555; ApplMicrobiolBiotechnol. 2013, 97(2): 719-729.), but there is no report on the use of this promoter in Trichosporium dermatosa and other specialties outside the Basidiomycota, such as Ascomycota spp.

Method used

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  • Promoter and terminator of 3-phosphoglyceraldehyde dehydrogenase and application thereof
  • Promoter and terminator of 3-phosphoglyceraldehyde dehydrogenase and application thereof
  • Promoter and terminator of 3-phosphoglyceraldehyde dehydrogenase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Extraction of total RNA from Lipomycessstarkeyi NRRLY-11557

[0073] L. starkeyi NRRLY-11557 (purchased from the American Agricultural Research Culture Collection (NRRL), Bacterial Foodborne Pathogens & Mycology Research, 1815N.University Street IL61604.Peoria, Illinois) was inoculated into 50ml YEPD liquid medium (glucose 20.0g / l, yeast extract 10.0g / l, peptone 20.0g / l, pH6.0), 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 cultured on a shaker at 30°C for 12 hours to reach the logarithmic growth phase. Centrifuge at 5000 rpm for 4 minutes at 4°C to collect the cells, freeze the cells quickly with liquid nitrogen, and grind to break the wall. Total RNA was extracted using the TakaRa RNAiso kit and following its standard procedures.

[0074] The RNA was subjected to 1.5% agarose gel electrophoresis, observed and identified using a fluorescence-ultraviolet analyzer, and...

Embodiment 2

[0075] Example 2: Synthesis of first strand of L. stardii NRRLY-11557cDNA and degenerate PCR of GPD

[0076] The first-strand cDNA was synthesized by reverse transcription using the total RNA of L. stariae NRRLY-11557 as a template. First, 1.0 μl total RNA (about 2 μg), 1.0 μl primer SMARTIV: 5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3′ and 1.0 μl ligodT-linker primer CDSIII / 3′: 5′-ATTCTAGAGGCCGAGGC2GGCCGACATG-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 a PCR tube and mixed evenly, kept at 72°C for 2 min, immediately placed on ice for 2 min, and 2.0 μl of 5 ×The first strand buffer (Clontech Company), 1.0 μl DTT (20 mM), 1.0 μl IdNTP (10 mM), 1.0 μl powerscript reverse transcriptase (Clontech Company) 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.

[0077] Design and synthesize two deg...

Embodiment 3

[0078] Example 3: Amplification of L. studleyi NRRLY-11557 Genomic DNA

[0079] Genomic DNA of L. stariae NRRLY-11557 was extracted by glass bead wall breaking method (Chapter 13 of the third edition of the Molecular Biology Experiment Guide, Osper et al., translated by Yan Ziying et al., published by Science Press). The prepared genomic DNA was measured by Nanodrop1000, and the OD was measured 260 / OD 280 =1.85, indicating that the quality of genomic DNA is very good. The concentration was 120ng / μl, 500μl in total, and the genomic DNA samples were frozen at -20°C for later use.

[0080] According to the cDNA sequence of glyceraldehyde phosphate dehydrogenase obtained in Example 2, a pair of gene-specific primers were designed, GPD-p1: 5'-ATGCTTAACCTCAAAGTATCTGTT-3' and GPD-p2: 5'-TTAGAACTTCTGCTCGACATCT-3', Using the genomic DNA of L. studlii NRRLY-11557 as a template, PCR amplification was performed according to conventional methods to obtain a PCR product of about 1.6 kb ...

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Abstract

The invention provides a promoter and terminator of 3-phosphoglyceraldehyde dehydrogenase. The promoter and terminator are obtained by amplifying the upstream and downstream sequences of the genomic DNA of 3-phosphoglyceraldehyde dehydrogenase of Lipomyces starkeyi and carrying out biological information analysis and function verification and can be widely used for gene expression, genetic engineering manipulation and strain improvement of Lipomyces and Trichosporon among Oleaginous yeasts. The nucleotide sequences of the promoter and terminator are SEQ ID No. 1 and SEQ ID No. 2, respectively. The invention also relates to DNA expression cassettes or recombinant vectors containing the promoter and terminator, and methods and corresponding bacterial strains used for constructing Lipomyces or Trichosporon gene engineering bacterial strains by using the promoter and terminator.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a promoter terminator of Lipomycessstarkeyi 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 deficien...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/70C12N15/81C12N1/21C12N1/19C12R1/645
Inventor 赵宗保林心萍张素芳王雪颖
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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