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CectGPDH2 (cytosolic glycerol-3-phosphate dehydrogenase 2) gene and application thereof

A gene and amino acid technology, applied in the field of glycerol-3-phosphate dehydrogenase gene CectGPDH2, can solve the problems of increased expression of ctGPDH and glycerol accumulation in cells

Inactive Publication Date: 2017-03-08
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

Under a certain range of osmotic stress, the expression of ctGPDH in cells will increase, which will lead to the accumulation of glycerol
[0009] At present, there is no report that the ctGPDH gene derived from plants can greatly increase the oil content of plants and increase the content of linoleic acid (C18:2) and eicosenoic acid (C20:1) in plants

Method used

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  • CectGPDH2 (cytosolic glycerol-3-phosphate dehydrogenase 2) gene and application thereof
  • CectGPDH2 (cytosolic glycerol-3-phosphate dehydrogenase 2) gene and application thereof
  • CectGPDH2 (cytosolic glycerol-3-phosphate dehydrogenase 2) gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Obtaining the full-length cDNA of Chlorella ellipsoidal CectGPDH gene

[0037] 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).

[0038] The cDNA synthesis reaction conditions are as follows:

[0039]

[0040]

[0041] RNase Free H 2 O to a total volume of 20 μL. After flicking to mix well and centrifuging briefly, carry out the reverse transcription reaction according to the following procedure: 30°C for 10 minutes, 42°C for 30 minutes,...

Embodiment 2

[0045] Embodiment 2 Contains the construction of the plant expression vector of CectGPDH2 gene

[0046] The plant expression vector pCAMBIA2301 from CAMBIA Company was selected as the plant expression vector of CeGPDH gene. Since there are no promoters and terminators with proper orientation near the multiple cloning site region of the pCAMBIA2301 vector, the vector pT305R with a promoter and terminator is selected as the intermediate vector (pUC18 vector is used as the backbone, self-built in the laboratory, ampicillin resistance), connect the CeGPDH2 gene to the multiple cloning site region between the promoter and the terminator on pT305R, and then select the upstream of the promoter and the downstream restriction sites of the terminator to cut off the complete gene with the promoter-CectGPDH-terminator Insert the sequence into the multiple cloning site region of the pCAMBIA2301 vector to complete the construction of the CeGPDH-2301 plant expression vector. The vector map i...

Embodiment 3

[0047] Embodiment 3 plant expression vector CeGPDH-2301 transforms Agrobacterium

[0048] Put 50 μL of fresh Agrobacterium competent cells on ice, add 1 μg of the plasmid DNA prepared in Example 2, which is the CeGPDH-2301 plant expression vector, mix well and place on ice for 30 min. Quickly freeze in liquid nitrogen for 1 min and then quickly transfer to a 37°C water bath for 5 min. Add 1mL of YEP liquid medium without antibiotics, and incubate at 28°C and 230rpm for 2-4h. Centrifuge at 3,000 rpm for 2 minutes to collect the bacteria, leave 100 μL of liquid to resuspend the bacteria, spread on YEP solid medium (containing 50 μL / 100 mL of kan, 30 μL / 100 mL of rif), and incubate at 28°C for 24-48 hours.

[0049] After 24-48 hours, colonies were picked from the culture medium plate and verified by colony PCR. Finally, the confirmed positive colonies were saved.

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Abstract

The invention provides a CectGPDH2 gene and an application thereof in increase of fatty acid content. The nucleotide sequence of the CectGPDH2 gene is shown as SEQ NO.1. The gene comes from Chlorella ellipsoidea and encodes GPDH, wherein the GPDH participates in shuttle of the mitochondrion G3P (glycerol-3-phosphate), provides electrons for respiratory chains, is a key enzyme for G3P synthesis, is one of the key enzymes for connecting glucose metabolism with lipid metabolism and plays an important role in lipid synthesis and energy metabolism in plants. The total fatty acid content of seeds of Arabidopsis thaliana and rape genetically modified with the gene can be increased obviously, and the gene can be used for increasing the oil content of plant cells and the content of linoleic acid (C18:2) and eicosenoic acid (C20:1) in the plants and has good application prospect.

Description

technical field [0001] The invention relates to a glycerol-3-phosphate dehydrogenase gene CectGPDH2 and its application. Specifically, it relates to the acquisition of the gene sequence and the construction of the plant expression vector, as well as its application to significantly increase the fatty acid content of the model plant Arabidopsis and oil crop seeds. 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 pote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/82A01H5/00
CPCC12N9/0006C12N15/8247C12Y101/01008
Inventor 胡赞民范成明陈宇红张丹李帅
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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