A method for improving carbon fixation efficiency of microalgae and transgenic Chlamydomonas and application

A transgenic, high-efficiency technology, applied in the field of biological genetic engineering, to achieve the effects of increasing growth rate, accelerating growth rate, and improving carbon sequestration efficiency

Active Publication Date: 2021-11-30
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] For the research results of recent researchers, we selected the chloroplast type by considering the characteristics of the Calvin cycle and the specific needs of the practical application of engineered microalgae (in most cases, biomass / carbon sequestration is the basis of downstream products). GAP3, a key enzyme involved in glycolysis in the cytoplasm, isoenzyme exists in the chloroplast and cytoplasm, and its product is also the key product of chloroplast carbon fixation GAP3, but few reports use it as a target to enhance carbon fixation

Method used

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  • A method for improving carbon fixation efficiency of microalgae and transgenic Chlamydomonas and application
  • A method for improving carbon fixation efficiency of microalgae and transgenic Chlamydomonas and application
  • A method for improving carbon fixation efficiency of microalgae and transgenic Chlamydomonas and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Transformation of the foreign gene corresponding to the GAP3 gene in Chlamydomonas reinhardtii and construction of the expression vector

[0059] The algal strain selected in this example is Chlamydomonas reinhardtii cc-137 (purchased from the American Chlamydomonas Center) as the recipient of the transgenic operation, and the algal strain is a wild-type Chlamydomonas reinhardtii strain.

[0060] The medium used in the cultivation of Chlamydomonas reinhardtii is TAP medium, and the formula of TAP medium is as follows:

[0061] Mother liquor 1 (salt solution): 20gNH 4 Cl, 5g MgSO 4 ·7H 2 O, 2.5g CaCl 2 2H 2 O is fixed in 500ml deionized water;

[0062] Mother liquor 2 (phosphate solution): 10.8gK 2 HPO 4 , 5.6gKH 2 PO 4 Set the volume to 500ml deionized water;

[0063] Mother liquor 3 (Hutner's trace metal salt solution): 50.0gNa 2 -EDTA·2H 2 O,22gZnSO 4 ·7H 2 O, 5.06gMnCl 2 4H 2 O, 1.61g CoCl 2 ·6H 2 O, 1.57g CuSO 4 ·5H 2O, 1.10g (NH 4 ) ...

Embodiment 2

[0074] Example 2: Genetic transformation of Chlamydomonas reinhardtii

[0075] The recombinant plasmid pChlamy-c-GAP3 was extracted by conventional SDS alkaline lysis method

[0076] The specific steps of the "electrotransfer method" are as follows:

[0077] (1) Cultivate wild-type Chlamydomonas reinhardtii cc-137 (Chlamydomonas Resource Center CC-137) in continuous light and TAP medium to OD=0.4-0.6, and dilute the cells to OD=0.05-0.08. After 24 hours of culture, algae When the liquid concentration reaches 0.3-0.6, take 15ml of algae cells at 2500rpm, centrifuge at room temperature for 10min, and discard the supernatant.

[0078] (2) Resuspend the algae cells with 250 μl of 40 mM sucrose TAP solution, add to the electric shock cup, and then add 1 to 2 μg of linearized pChlamy-c-GAP3 plasmid to the cup for electric shock (600V, 50uF, infinite resistance).

[0079] (3) After the electric shock, the mixture of algal cells and exogenous DNA in the electric shock cup was added ...

Embodiment 3

[0082] Example 3: Screening and identification of transgenic Chlamydomonas reinhardtii

[0083] The expression vector of C. reinhardtii contains Hygromycin resistance gene, so it has hygromycin resistance, and the successfully transformed algal strains can grow on the plate containing hygromycin resistance. The detection of transgenic algae includes PCR verification of single algae colony at the gene level and western blotting verification at the protein level.

[0084] (1) Double PCR verification of the single algal fall of the transformed strain at the gene level

[0085] Pick the single algae on the plate and drop it into the PCR tube, add 10 μl of sterile water to resuspend the algae cells, cook at 98°C for 20-30 minutes, centrifuge to get the supernatant, use PC-F / R primers, (Use PrimeSTAR Max DNA Polymerase , TaKaRa, Dalian City, CodeNo.R045Q), using PCR technology to amplify the target gene fragment from the algae, and then using the GAP3-specific primer (GAP3-F / R) on ...

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Abstract

The invention discloses a method for improving the carbon fixation efficiency of microalgae. Chlamydomonas chloroplast-type glyceraldehyde-3-phosphate dehydrogenase gene was overexpressed to enhance the ability of Chlamydomonas photosynthetic carbon fixation. It specifically includes the construction of a glyceraldehyde-3-phosphate dehydrogenase gene recombinant expression vector, and transforming the glyceraldehyde-3-phosphate dehydrogenase gene recombinant expression vector into Chlamydomonas reinhardtii cells by electric shock transformation, and screening to obtain glyceraldehyde-3 ‑Phosphate dehydrogenase enhanced expression in transgenic Chlamydomonas reinhardtii. Microalgae can synthesize biomass such as oil, protein, starch, and carotenoids through photosynthetic carbon fixation, and accelerating the production efficiency of microalgal biomass plays an important role in downstream industries. The invention transfers the target gene into Chlamydomonas reinhardtii, accelerates the carbon fixation efficiency and increases its growth rate, and has significant application prospects in the field of microalgae bioengineering.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and relates to a method for transferring a target gene into the genome of Chlamydomonas reinhardtii and accelerating the growth rate of Chlamydomonas reinhardtii, in particular to a transgenic Chlamydomonas reinhardtii for improving the carbon fixation effect of Chlamydomonas reinhardtii, Build methods and their uses. Background technique [0002] With the development of human society, the advancement of industrialization and the increase of population, CO 2 emissions are increasing and the greenhouse effect is becoming more and more serious; therefore, reducing CO 2 The emission and accumulation of carbon dioxide are the focus of solving global warming. The photosynthesis in the life process of organisms completes the biological carbon sequestration, which is the way to realize the carbon cycle in nature. However, trees, plants can only slowly absorb CO from the atmosph...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/13C12N15/80C12R1/89
Inventor 薛松朱振曹旭鹏苑广泽
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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