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Cem GPDH (Glycerol-3-phosphate dehydrogenase) gene and application thereof

A gene and amino acid technology, applied in the field of glycerol-3-phosphate dehydrogenase gene GPDH, can solve the problem of less research on key enzyme genes

Active Publication Date: 2015-02-18
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Microalgae have strong reproductive ability, low requirements on the growth environment, easy cultivation, high photosynthetic efficiency, and high oil content. However, there are relatively few studies on the key enzyme genes in the lipid metabolism pathway of microalgae. [1-2]

Method used

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  • Cem GPDH (Glycerol-3-phosphate dehydrogenase) gene and application thereof
  • Cem GPDH (Glycerol-3-phosphate dehydrogenase) gene and application thereof
  • Cem GPDH (Glycerol-3-phosphate dehydrogenase) gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1. Obtaining the full-length cDNA of Chlorella ellipsoidal CemGPDH gene

[0027] Take the algae liquid of Chlorella ellipsoides (from the Institute of Hydrobiology, Chinese Academy of Sciences) in logarithmic growth phase that has been cultured for 3-4 days, collect the algae bodies by centrifugation, and place them in liquid nitrogen to grind them thoroughly. Total RNA was extracted and purified according to the instructions of Aidelai EASYspin RNA Extraction Kit and the instructions of Takara DNase I. cDNA was obtained by reverse transcription with polyT primers, and RT-PCR was performed (see TOYOBO ReverTra Ace-α Kit for specific operations).

[0028] The cDNA synthesis reaction conditions are as follows:

[0029]

[0030] RNase Free H 2 O to a total volume of 20 μL. After flicking to mix well and centrifuging briefly, perform the reverse transcription reaction as follows:

[0031]

[0032] An appropriate amount of the above-mentioned reverse transcri...

Embodiment 2

[0036] Embodiment 2. Construction of the yeast expression vector containing CemGPDH gene

[0037] According to the cds sequence of CemGPDH, primers with KpnI and EcoR I restriction sites were designed (the sequences of the two primers were: cgccGGTACCATGAAGTTGTCAAAGATTTAC and ccggGAATTCCTATGTCTTGACTGGGGTC from the pEASY-Blunt vector containing CemGPDH, using high-fidelity EasyPfu DNA Polymerase (purchased from Transgen Company), amplified the fragment containing KpnI and EcoR I restriction sites, carried out double restriction digestion with KpnI and EcoR I (purchased from Takara Company), and yeast expression vector pYES2 (purchased from Invitrogen Company) with the same double restriction enzyme ) connection, sequencing verification, and named it pYES-GPDH, its vector diagram is shown in figure 1 .

Embodiment 3

[0038] Example 3. Transformation of Saccharomyces cerevisiae with yeast expression vector pYES-GPDH

[0039] Inoculate uracil-deficient Saccharomyces cerevisiae INVSC1 (purchased from Invitrogen) in 10 mL of LYPD medium, and culture overnight at 30°C with shaking. Inoculate the bacterial solution into 50mL YPD medium the next day and dilute to OD 600 =0.4, continue to culture for 2-4h to OD 600 Between 0.4-0.6, 5,000rpm refrigerated centrifugation for 1min, with 40mL 1 × TE (10mM Tris, pH7.5, 1mM EDTA) to suspend the precipitate, 5,000rpm refrigerated centrifugation, with 2mL 1 × LiAc (10mM lithium acetate, pH7.5) / Suspend the pellet in 0.5×TE and incubate at room temperature for 10 min. Mix 100 μL of yeast suspension with 1 μg of yeast expression vector pYES-GPDH and 100 μg of denatured salmon sperm DNA, then add 700 μL of 1×LiAc / 40% PEG-3350 / 1×TE, and mix well. Incubate at 30°C for 30 minutes, add 88 μL DMSO, mix well, and heat shock at 42°C for 7 minutes. Centrifuge at...

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Abstract

The invention provides a Cem GPDH (Glycerol-3-phosphate dehydrogenase) gene and an application of the Cem GPDH gene in the aspect of increase of cellular fatty acid content. A nucleotide sequence of the Cem GPDH gene is represented as SEQ ID NO: 1. The gene is derived from Chlorella ellipsoidea, and GPDH is coded. G3P (Glycerol-3-phosphate) synthesized through catalysis by GPDH is an important raw material for synthesis of TAG (triacylglycerol), the enzyme participates in mitochondria G3P shuttle, provides electrons for a respiratory chain, is a key enzyme for G3P synthesis, is one of key enzymes for connecting glycometabolism and lipid metabolism and has an important effect on lipid synthesis and energy metabolism in plants; and besides, the gene can remarkably increase the total fatty acid content of cells when utilized to convert yeast cells, plant cells and microalgae cells.

Description

technical field [0001] The invention relates to a glycerol-3-phosphate dehydrogenase gene GPDH and its application. Specifically, it relates to the acquisition of the gene sequence, the construction of the yeast expression vector, and its use in significantly increasing the fatty acid content of yeast. Background technique [0002] At present, with the excessive consumption of traditional energy and the increasing energy demand, the energy problem is becoming more and more serious, and the research and development of alternative energy has become a hot spot that scholars around the world pay attention to. Biodiesel is mainly processed into liquid fuels from oil crops such as soybeans and jatropha curcas, waste kitchen fats, animal fats, and genetically engineered microalgae. It is a high-quality new renewable energy that can replace traditional petroleum fuels. Microalgae are considered to be one of the most potential energy resources due to their high oil content, easy cul...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/81C12N15/82C12N15/79C12N1/19C12N1/13C12N5/10C12P7/64C12R1/865C12R1/89
CPCC12N9/0006C12P7/649C12Y101/05003Y02E50/10
Inventor 胡赞民张丹李帅范成明陈宇红
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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