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3-phosphoglyceric kinase promoter and terminator and applications thereof

A phosphoglycerate kinase and promoter technology, which is applied in the field of genetic engineering, can solve the problem that the promoter sequence report of T. dermoides has not been seen, that it is not applicable to lipid yeast and T. stipitis, restricts strain modification, etc. problems, to achieve the effect of promoting strain improvement and metabolic engineering research

Active Publication Date: 2016-07-20
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • 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 the phosphoglycerate kinase promoter of rhizopus oryzae (Rhizopusoryzae) and arbuscular mycorrhizal fungus (Glomusmosseae) was once isolated and used for genetic engineering of filamentous fungi (mold) (US6528636; US6465635; Harrier, LA1., Paterson, LJ.CurrGenet, 2002, 42 (3): 169-178.), but these promoters are all unsuitable for Lipomyces starburi and Trichosporium dermatitis

Method used

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  • 3-phosphoglyceric kinase promoter and terminator and applications thereof
  • 3-phosphoglyceric kinase promoter and terminator and applications thereof
  • 3-phosphoglyceric kinase promoter and terminator and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1: Extraction of total RNA from Lipomycessstarkeyi NRRLY-11557

[0088] L. starkeyi NRRLY-11557 (purchased from the American Agricultural Research Culture Collection (NRRL), Bacterial Foodbome 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.

[0089] The RNA was subjected to 1.5% agarose gel electrophoresis, observed and identified using a fluorescence-ultraviolet analyzer, and ...

Embodiment 2

[0090] Example 2: First-strand synthesis of L. stardii NRRLY-11557 cDNA and PGK degenerate PCR

[0091] 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′-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 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.

[0092] Design and synthesize two degenerat...

Embodiment 3

[0093] Example 3: Amplification of L. studleyi NRRLY-11557 Genomic DNA

[0094] 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 future use.

[0095] According to the 3-phosphoglycerate kinase cDNA sequence obtained in Example 2, design a pair of gene-specific primers, PGK-p1: 5'-ATGCTTAACCTCAAAGTATCTGTT-3' and PGK-p2: 5'-TTAGAACTTCTGCTCGACATCT-3', Es Genomic DNA of L. dablii NRRLY-11557 was used as a template, PCR amplification was performed according to conventional methods, and a PCR product of about 1.6 kb was obtained (not shown). ...

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Abstract

Through amplifying lipomyces starkeyi 3-phosphoglyceric kinase genome DNA upstream and downstream sequences and carrying out biology information analysis and functional verification, a promoter and terminator that can be widely applied to gene expression, genetic engineering operation, and strain improvement of Lipomyces and Trichosporon can be obtained. The nucleotide sequence of the promoter and terminator are represented by the SEQ ID No.1 and SEQ ID No.2. The invention also relates to a DNA expression cassette or recombinant carrier comprising the elements, a method using related elements to construct gene engineering strains of Lipomyces or Trichosporon, 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 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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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/70C12N15/81C12N1/21C12N1/19
Inventor 赵宗保林心萍张素芳王雅南
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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