A kind of transgenic Chlamydomonas for improving fatty acid content of Chlamydomonas reinhardtii, construction method and use thereof

A technology of Chlamydomonas reinhardtii and transgene, which is applied in the field of constructing and increasing the fatty acid content of Chlamydomonas reinhardtii, and can solve problems such as the transformation and expression of Chlamydomonas reinhardtii and the influence of fatty acid synthesis of Chlamydomonas reinhardtii.

Active Publication Date: 2017-10-17
SHENZHEN UNIV
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  • Abstract
  • Description
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Problems solved by technology

Although LPAAT and DAGAT have important functions in TAG synthesis, there are few researches on the function of LPAAT and DAGAT in Chlamydomonas reinhardtii at home and abroad, and there is no research on their transformation and expression in Chlamydomonas reinhardtii, let alone their effect on Effects of Chlamydomonas reinhardtii on Fatty Acid Synthesis

Method used

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  • A kind of transgenic Chlamydomonas for improving fatty acid content of Chlamydomonas reinhardtii, construction method and use thereof
  • A kind of transgenic Chlamydomonas for improving fatty acid content of Chlamydomonas reinhardtii, construction method and use thereof
  • A kind of transgenic Chlamydomonas for improving fatty acid content of Chlamydomonas reinhardtii, construction method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Transformation of lpaat and gpd1 gene corresponding exogenous genes and construction of expression vectors in Chlamydomonas reinhardtii

[0064] The algal strain selected in this example is Chlamydomonas reinhardti CC-849 (purchased from the Chlamydomonas reinhardti CC-849) (purchased from the American Chlamydomonas species bank, Durham, NC27708 USA) as the recipient of the transgenic operation, and the algal strain is cell wall-deficient strains of Chlamydomonas reinhardtii.

[0065] The medium used for the cultivation of Chlamydomonas reinhardtii is TAP medium, and the formulation of 1L medium is as follows: 2.42g Tris, 25mL 4 × Beijerinck salts (16g NH 4 Cl, 2g CaCl 2 .2H 2 O,4g MgSO 4 .7H 2 O was dissolved in water, and the volume was adjusted to 1L), 1mL 1M potassium phosphate buffer solution, 1mL Trace trace element mixed solution (11.4g H 3 BO 3 ,5.6g MnCl 2 .4H 2 O,22gZnSO 4 .7H 2 O, 4.99g FeSO 4 .7H 2 O, 1.61g CoCl 2 .6H 2 O, 1.57g CuSO...

Embodiment 2

[0081] Example 2: Genetic transformation of Chlamydomonas reinhardtii

[0082] 1) "Bead milling method" genetic transformation

[0083] use Plasmid Purification Kit ( Germany) to extract the recombinant plasmids pH-c-lpaat and pH-c-gpd1 respectively.

[0084] The specific steps of the "bead milling method" are as follows: (1) Cultivate the cell wall-defective Chlamydomonas reinhardtii CC-849 (purchased from the American Chlamydomonas species bank) to the logarithmic phase in continuous light and TAP culture medium, and the number of cells is about 1-2×10 6 cells / ml. Centrifuge at room temperature at 5000rmp for 5min to collect algae cells; discard the supernatant;

[0085] (2) Resuspend the algae cell pellet with sterilized fresh TAP culture medium, and adjust the cell concentration to 2×10 8 cells / ml;

[0086] (3) Draw 270μl of algae cell suspension into a 1.5ml EP tube (with sterilized alloy tin beads inside), and then add 1μg (for separate transformation, pH-c-lpa...

Embodiment 4

[0097] Example 4: Screening and identification of transgenic Chlamydomonas reinhardtii

[0098] The expression vector of Chlamydomonas reinhardtii contains ble gene, so it has bleomycin resistance, and the transformed strain can grow on the plate containing bleomycin resistance. The detection of transgenic algae includes PCR sequencing, RT-PCR analysis and determination of fatty acid content.

[0099] (1) RT-PCR

[0100] Extract the green monoclonal Chlamydomonas reinhardtii total RNA of Example 3, take 1ug for reverse transcription (60 minutes at 42°C, 15 minutes at 72°C) (use TaKaRa reverse kit, Takara, Dalian, Code No.2641A) The cDNA was obtained, and the target gene fragments (c-lpaat and c-gpd1) and the internal reference gene (actin gene) were amplified from the cDNA by PCR technology, and the PCR amplification products were sent to Shanghai Sangong Bioengineering Company for sequencing. In the embodiment, actin gene primer 3: 5'acccgtgctgctgactg 3' is SEQ ID NO: 11 an...

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Abstract

The invention discloses a transgenic Chlamydomonas for improving fatty acid content of Chlamydomonas reinhardtii, as well as a construction method and application thereof. The construction method comprises the following steps: respectively constructing a lysophosphatidic acid acyltransferase gene recombination expression vector and a glycerol 3-phosphate acyl transferase gene recombination expression vector; and respectively or jointly converting the lysophosphatidic acid acyltransferase gene recombination expression vector and the glycerol 3-phosphate acyl transferase gene recombination expression vector to the Chlamydomonas reinhardtii cell, thereby obtaining converted lysophosphatidic acid acyltransferase gene and / or glycerol 3-phosphate acyl transferase gene Chlamydomonas reinhardtii. According to the method, exogenous genes are converted into the Chlamydomonas reinhardtii, thus, oil production of the Chlamydomonas reinhardtii is increased, compared with the physicochemical methods, such as mutagenesis, effects of the method provided by the invention are more remarkable, and the Chlamydomonas reinhardtii has genetic stability; the obtained transgenic Chlamydomonas has increased oil production and lowered polyunsaturated fatty acids proportion, and saturation and modification steps in a biodiesel production process are reduced.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and relates to a method for optimizing and transforming exogenous genes into the genome of Chlamydomonas reinhardtii and increasing the fatty acid content of Chlamydomonas reinhardtii, in particular to a transgenic Chlamydomonas reinhardtii for increasing the fatty acid content of Chlamydomonas reinhardtii , construction methods and their uses. Background technique [0002] At present, about 80% of the world's energy comes from fossil fuels. As a non-renewable energy source, fossil fuels are gradually exhausted, and their depletion has become a major problem facing people. Pollution, health and other issues. The use of fossil fuels is one of the main causes of global warming. As a source of energy, fossil fuels should be replaced by a renewable and clean energy source in order to reduce greenhouse gas emissions. Long-term dependence on fossil fuels is not conducive to lon...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/13C12N15/54C12N15/63C12R1/89
CPCC12N9/1029C12N15/79C12Y203/01015C12Y203/01023
Inventor 王潮岗胡章立李逸
Owner SHENZHEN UNIV
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