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Gene and protein related to lipid metabolism pathway of chlamydomonas reinhardtii, protein, gene mutation strains and applications thereof

A metabolic pathway, the technique of Chlamydomonas reinhardtii, applied in changing the oil content of Chlamydomonas reinhardtii, in the field of mutant algae strains, can solve the problems of slow progress, no new reports on synthesis and regulation pathways, and weaknesses

Inactive Publication Date: 2014-04-02
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

At present, the research on the molecular mechanism of oil synthesis and metabolism in microalgae is still very weak, and little is known about the functions of genes related to oil synthesis and accumulation network pathways in microalgae. The known functional genes related to fatty acid synthesis in plant cells include acetyl-CoA carboxylation enzyme (acetyl-coenzyme A carboxylase, ACCase) gene, glycerol triphosphate dehydrogenase gene (g3pdh), diacylglycerol fatty acyltransferase (dagat) gene, etc. algae to obtain high-oil algae strains that highly express the ACCase gene, but they have not been successful. At present, international research on the use of genetic engineering methods to increase the oil content of algae is also progressing slowly.
Although some genes related to the oil synthesis pathway in plants have homologous genes in microalgae, there are no new reports on whether the oil synthesis pathway is the same as in plants, and how the synthesis and regulation pathways are in microalgae

Method used

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  • Gene and protein related to lipid metabolism pathway of chlamydomonas reinhardtii, protein, gene mutation strains and applications thereof
  • Gene and protein related to lipid metabolism pathway of chlamydomonas reinhardtii, protein, gene mutation strains and applications thereof
  • Gene and protein related to lipid metabolism pathway of chlamydomonas reinhardtii, protein, gene mutation strains and applications thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1. Cloning of lipid metabolism pathway-related genes (AT, TAGLP) in Chlamydomonas reinhardtii

[0082] The inventors found in the research that there are more than 2,500 genes in Chlamydomonas reinhardtii whose differential expression in the four growth stages is more than twice, and they play different roles in regulating the decomposition or synthesis of lipids. In the in-depth research, the inventor screened out genes related to the lipid metabolism pathway (such as AT, TAGLP, LPOGA, G3PDH, LPAAT and DAGAT six genes, the genes in the text are in italics, the same below), among which the AT-encoded protein AT is the acetyl transfer Enzyme, which can catalyze the reaction of acetyl coenzyme A and long-chain alcohol to form coenzyme A and long-chain ester; TAGLP encoding protein TAGLP is a triglyceride lipase, which can reduce triacylglycerol to diacylglycerol and fatty acid anion, from glycerol The reverse reaction for the synthesis of triacylglycerols. The pr...

Embodiment 2

[0085] Embodiment 2, the RNA (microRNA) that suppresses AT, TAGLP gene expression and the acquisition of coding sequence thereof, sequence analysis and the construction of AT, TAGLP gene silencing vector

[0086] 1. Acquisition of RNA (microRNA) for silencing AT gene and its reverse complementary sequence coding gene

[0087] According to the AT gene sequence, according to the principle of microRNA design (WMD3-Web MicroRNA Designer (http: / / wmd3.weigelworld.org / cgi-bin / webapp.cgi)), a target site was finally selected, which is located in the AT gene mRNA sequence transcription A sequence of 21bp in length downstream of the start site aug, the obtained microRNA that inhibits AT gene expression is named AT-miR, AT-miR: 5'-GAGUCGCAUAUGCUGUUAUUA-3', AT-miR acts on AT mRNA 96-115 position sequence 5'-GAGUCGCAUAUGCUGUUAUUA-3'. Then obtain its coding sequence and the coding gene capable of synthesizing the RNA (microRNA) and its reverse complementary sequence according to the AT-miR...

Embodiment 3

[0143] Embodiment 3, the acquisition of Chlamydomonas reinhardtii mutant strain AT-CR and TAGLP-CR

[0144] 1. Transforming AT and TAGLP gene silencing vectors into Chlamydomonas reinhardtii

[0145] The AT gene silencing vector AT-mic-RNA2 carrying AT-miDNA and the TAGLP gene silencing vector TAGLP-mic-RNA2 carrying TAGLP-miDNA were transformed into Chlamdomonas reinhardtii CC425 and Chlamdomonas reinhardtii (from Beijing Life Sciences) respectively. Institute of Science) is an arginine auxotrophic algal strain that cannot synthesize arginine (Arg) by itself, while the silencing vectors AT-mic-RNA2 and TAGLP-mic-RNA2 corresponding to AT-miDNA and TAGLP-miDNA both contain Amp Resistance gene and Arg gene, so if a single clone grows on the arginine-free plate after transformation, it means that the transformation is successful. like image 3 As shown, the specific conversion method is as follows:

[0146] (1) Carry out single enzyme digestion of the plasmids AT-mic-RNA2 and ...

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Abstract

The invention discloses gene and protein related to lipid metabolism pathway of chlamydomonas reinhardtii, protein, gene mutation strains and applications thereof. According to the invention, silent treatment is carried out respectively on the two genes by designing microRNA(AT-mic and TAGLP-mic) corresponding to the two genes AT and TAGLP, and silent mutation strains (AT-CR and TAGLP-CR) of the AT and TAGLP genes of the chlamydomonas reinhardtii are obtained; by determination on the contents of neutral lipids and the total contents of lipids of the mutation strains, the contents of the neutral lipids of the mutation strains AT-CR and TAGLP-CR are respectively increased by 14.73% and 13.18% as compared with the wild type, and the contents of the total lipids of the mutation strains AT-CR and TAGLP-CR are respectively increased by 76.97% and 102.03% as compared with the wild type; the invention plays an important role in extracting the contents of the lipids from the chlamydomonas reinhardtii, and is wide in application prospect.

Description

technical field [0001] The invention belongs to the field of genetic engineering and microorganism technology, and in particular relates to two genes (AT, TAGLP) related to the lipid metabolism pathway of Chlamydomonas reinhardtii and the mutant algae strains of these two genes, and their use in changing the oil content of Chlamydomonas reinhardtii Applications. Background technique [0002] As a strategic reserve of bioenergy, microalgae energy has attracted great attention at home and abroad. Microalgae has the advantages of not competing with crops for land, no pollution, easy cultivation, rapid reproduction, and high oil content, so it is considered to be an important raw material for the sustainable development of bioenergy today. At present, the research on microalgae with high oil production is mainly focused on screening from germplasm banks, artificial mutagenesis and using genetic engineering and metabolic engineering to manufacture engineered algae strains. Compa...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/10C12N15/55C12N9/20C12N15/113C12N15/79C12N1/13C12R1/89
Inventor 田朝光齐西珍卢丽娜吕和鑫曲戈马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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