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Method for increasing the content of total oil, linoleic acid or α-linolenic acid in chlorella

A technology of chlorella and linoleic acid, applied in the direction of microorganism-based methods, biochemical equipment and methods, and the use of vectors to introduce foreign genetic materials, etc., can solve the problems of seed germination and leaf growth defects, which have not yet been retrieved

Inactive Publication Date: 2011-12-14
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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Problems solved by technology

Shen et al. placed the maize ZwLEC1 gene under the embryo-specific weak promoter EPA1, and obtained transgenic maize with high oil content, but the plant leaves were reduced to about half of the wild type; placed the maize ZwLEC1 gene under the embryo-specific strong promoter. Under the promoter OLE, very severe defects in seed germination and leaf growth occur [12]
At present, no research has been found on the use of the LEC1 gene encoded by Arabidopsis to transform algae and increase oil content

Method used

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  • Method for increasing the content of total oil, linoleic acid or α-linolenic acid in chlorella
  • Method for increasing the content of total oil, linoleic acid or α-linolenic acid in chlorella
  • Method for increasing the content of total oil, linoleic acid or α-linolenic acid in chlorella

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Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1. Construction of AtLEC1 gene chlorella vector

[0038] 1. Synthesize the gene AtLEC1 according to the published sequence (NCBI Reference Sequence: NM 102046.4), and connect it into the T vector (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) to form the vector pEB-AtLEC1 containing the gene AtLEC1, and sequence the gene AtLEC1 The sequence is SEQ ID NO: 1, and the expressed protein sequence is SEQ ID NO: 2. The plasmid vector diagram is shown in figure 1 .

[0039] 2. Using pGreen0029 (purchased from the John Innes Centre) as the base vector, construct a control vector pG29-UN-CK containing Ubiquitin promoter and nos terminator.

[0040] Use high-fidelity enzyme Pfu (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) to amplify the nos terminator from the vector pGreen0029, with primers SEQ ID NO: 3 (forward primer) and SEQ ID NO: 4 (reverse primer) .

[0041] The PCR reaction system is as follows:

[0042]

[0043]

[0044] Th...

Embodiment 2

[0065] Example 2. Acquisition and detection of Chlorella transgenic AtLEC1

[0066] 1. Transformation of Chlorella

[0067] The chlorella used is Chlorella ellipsoidea (Chlorella ellipsoidea, from Institute of Hydrobiology, Chinese Academy of Sciences), refer to literature [2] Cultivate Chlorella to a suitable growth state, collect the algae cells by centrifugation and carry out hypertonic treatment, using the above literature [2] The same electric shock transformation condition carried out electric shock to Chlorella ellipsoides, and the Chlorella cells were coated with 30mg / L G418 solid medium containing 30mg / L G418 solid medium after electric shock (refer to literature [2] ), after about 4 to 6 weeks, detect the algae colonies grown on the plate.

[0068] 2. PCR detection of transgenic algal strains

[0069] Pick the single algal colony cells on the plate and inoculate them in 15mg / L G418 liquid medium (refer to literature [2] ) for 2 weeks (at a concentration of approx...

Embodiment 3

[0107] Example 3. Growth curve of Chlorella

[0108] In Example 2, the offspring LEC1-1 and LEC1-3 of the positive algal strains detected by molecular detection, and the control vector transformed algal strain CK, refer to the literature [2] Expand culture in 15mg / L G418 liquid medium, and wild algae (WT) expand culture in liquid medium without antibiotics at the same time. After culturing in a 50mL Erlenmeyer flask for 4-6 days, expand the culture in 150mL of new medium at an initial concentration of about 0.2g / L dry weight, sample and measure the biomass at the same time every day, and record for 8 consecutive days. The measurement results are shown in Table 1.

[0109] According to the measurement results, make the growth curves of transgenic algae and wild algae, see Figure 8 .

[0110] exist Figure 8 It can be found that Chlorella enters the plateau stage (growth reaches dynamic equilibrium) from the 6th day, and the algae biomass begins to decline on the 8th day. T...

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Abstract

The invention provides the use of an AtLEC1 gene in improvement on oil and fat content in chlorella. The gene is derived from Arabidopsis thaliana which is a member of CCAAT-Box binding factor HAP3 family. The AtLEC1 gene-transformed chlorella can improve the LA, oleic acid and ALA content and can improve the total oil and fat content of the chlorella strain. The method can be used in production of unsaturated fatty acid and biodiesel by an alga expression system and by using gene engineering technology.

Description

technical field [0001] The present invention relates to the application of plant coding gene in chlorella. Specifically, it involves constructing a gene AtLEC1 with a complete reading frame derived from Arabidopsis thaliana (Arabidopsis thaliana) into a Chlorella expression vector, and transforming it into Chlorella, so that the linoleic acid (LA) and α-linolenic acid (ALA) content, and total fat content were significantly increased. technical background [0002] The photosynthesis efficiency of microalgae is high, the growth cycle is short, the annual output per unit area is dozens or even hundreds of times that of grain, and the lipid content of microalgae is 10% to 70%, which is far beyond the reach of land plants. It has the advantages of large production capacity, no pollution, renewable, easy to cultivate, and contains more lipids. It grows and reproduces in waters, and will not cause conflicts with competition for agricultural and pastoral land. Due to the above ad...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12R1/89
Inventor 胡赞民张建辉白丽莉尹维波左建儒陈宇红宋丽英
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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