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Method for increasing lipid content of diatom through metabolism gene engineering

A technology for genetic engineering and oil content, applied in the field of genetic engineering, can solve problems such as unsuccessful, and achieve the effects of increasing oil content, promoting binding, and increasing diatom oil content

Inactive Publication Date: 2016-01-06
南通中国科学院海洋研究所海洋科学与技术研究发展中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Gene transfer is effective in raising triglyceride levels in canola and potatoes, but not in marine diatoms

Method used

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  • Method for increasing lipid content of diatom through metabolism gene engineering
  • Method for increasing lipid content of diatom through metabolism gene engineering
  • Method for increasing lipid content of diatom through metabolism gene engineering

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1, Cloning and functional verification of the glycerol-3-phosphate acyltransferase gene of Phaeodactylum tricornutum

[0038] 1. Cloning of cDNA sequence of Phaeodactylum tricornutum glycerol-3-phosphate acyltransferase gene

[0039] Centrifuge at 6000r / min for 5min to collect diatom cells, transfer to a pre-cooled mortar and grind with liquid nitrogen, extract RNA with RNApreppurePlantKit (TaKaRa, Dalian), detect RNA quality by 1.6% agarose gel electrophoresis, and measure OD 230 / OD 280 、OD 260 / OD280 Ratio, detection of RNA concentration and purity. Using PrimeScript1 st StrandcDNASynthesisKit (TaKaRa, Dalian) was used to perform reverse transcription to prepare cDNA. Set the reverse transcription system to incubate at 42°C for 30-60min, denature at 95°C for 5min, and store at 20°C for later use.

[0040] The mRNA sequence of the glycerol-3-phosphate acyltransferase gene of Phaeodactylum tricornutum (XM_002181618.1) has been annotated in GenBank, but its ...

Embodiment 2

[0063] Example 2: Introduction and detection of endogenous glycerol-3-phosphate acyltransferase gene in Phaeodactylum tricornutum by electric shock method

[0064] 1. Construction of diatom overexpression vector for Phaeodactylum tricornutum glycerol-3-phosphate acyltransferase gene

[0065] Using the yeast expression vector pYGPAT1 as a template, design a primer pair F3 (5'-CGAGCTCATGGCGATGCGAGCC-3') and R3 (5'-CGAGCTCTCACTTGGGTTTGTTTG-3') with a restriction endonuclease SacI recognition site, and glycerol-3-phosphate The acyltransferase gene was amplified and inserted into the self-built diatom nuclear expression vector pfcpA-MCS-fcpB-bar ( Figure 8 ) in the MCS (multiple cloning site) site, thereby constructing and completing the diatom overexpression vector pfcpA-gpat1-fcpB-bar ( Figure 9 ). Because it is inserted by SacI single enzyme digestion, in order to determine the direction of insertion, primer F4 (5'-AACCCCCACACGATTTTT-3') with one end positioned on the vector...

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Abstract

The invention provides a method for increasing the lipid content of diatom through metabolism gene engineering. A glycerinum-3-phosphate acyl transferase gene cDNA of phaeodactylum tricornutum which belongs to marine lipid-producing diatom is cloned, the function is verified in a glycerinum-3-phosphate acyl transferase gene mutant of yeast, a diatom overexpression carrier is established, phaeodactylum tricornutum cells are introduced with an electroporation method or a particle bombardment method, screening is performed through glufosinate, an engineered strain with increased TAG (triglyceride) is obtained and can be used for extracting microalgae lipid after being subjected to scale-up culture. Glycerinum-3-phosphate acyl transferase in the diatom is taken as a target regulated in diatom lipid metabolism gene engineering, the lipid content of the diatom is increased with the metabolism engineering method, and a new concept and a new focus are provided for lipid research of the diatom.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a method for increasing diatom oil content by introducing an endogenous glycerol-3-phosphate acyltransferase gene using metabolic genetic engineering technology. Background technique [0002] Biodiesel refers to the formation of mono-alkyl fatty acid esters through transesterification of oils (mainly triglycerides) from living organisms and alcohols (methanol or ethanol). The performance of biodiesel is basically equivalent to that of petrochemical diesel, and it has good environmental protection and biodegradability. It has become the most widely used and fastest growing ideal renewable energy in the world. [0003] Unicellular eukaryotic microalgae are photoautotrophic low-level aquatic plants with various types and wide distribution. They have the characteristics of high photosynthetic efficiency, fast growth and large biomass. Some microalgae from Chlorophyta and Diatoms hav...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/13C12N15/82C12N15/54C12P7/64
Inventor 姜鹏郭扬赵瑾陈华新
Owner 南通中国科学院海洋研究所海洋科学与技术研究发展中心
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