Protonema engineering blue algae with high fat content and high essential fatty acid content and constructed by adopting reverse vector method

A technology for essential fatty acids and filaments, which is applied in the fields of biotechnology and microalgae energy, can solve the problem of not being able to ultimately improve lipid yield, etc., and achieve the effects of high photosynthesis efficiency, high lipid yield, and pollution avoidance.

Active Publication Date: 2014-03-19
SHANGHAI OCEAN UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

These methods do not actually

Method used

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  • Protonema engineering blue algae with high fat content and high essential fatty acid content and constructed by adopting reverse vector method
  • Protonema engineering blue algae with high fat content and high essential fatty acid content and constructed by adopting reverse vector method
  • Protonema engineering blue algae with high fat content and high essential fatty acid content and constructed by adopting reverse vector method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Fishing out the target gene and constructing the vector

[0040] (1) Determine the pepc gene sequence, which can be found in GenBank, and its number is NP_488901; design primers according to this sequence, and use PCR technology to catch the pepc gene; the primer sequence is:

[0041] Upstream 5'-GATGGGCAAGCCACAAAAGACC-3', such as SEQ ID No.2;

[0042] Downstream 5'-AGGATCCGCGGGACGAGAAC-3', such as SEQ ID No.3;

[0043] Anabaena 7120 was purely cultured, and genomic DNA was extracted from it as a template for pepc gene, and the target gene was obtained by using PCR technology and the above primers, the length of which was about 2300bp, such as the sequence of SEQ ID No.1.

[0044] (2) Select the pRL-489 plasmid and connect it with the target gene to construct two kinds of shuttle expression vectors, forward and reverse. The forward vector is constructed according to the operation of gene overexpression vector construction in conventional genetic engineering, ...

Embodiment 2

[0052] Example 2 Transformation of cyanobacteria and screening of transformants.

[0053] This example includes:

[0054] (1) Purify and preserve the desired wild-type strain of cyanobacteria; (2) Determine the growth curve of the selected cyanobacteria, and cultivate the cyanobacteria to the mid-logarithmic growth stage, and use them as materials for the transformation of the three-parent conjugation transfer method; (3) Cultivate the Escherichia coli mutant carrying the above-mentioned shuttle expression vector to the mid-logarithmic growth phase (cultivation for 4 to 5 hours) for triparental conjugation transfer; ) of Escherichia coli was cultured to the mid-logarithmic growth phase (cultivation for about 4 to 5 hours), and used to transform cyanobacteria by the three-parent conjugative transfer method; (5) transform the cyanobacteria with the vector with pepc by the three-parent conjugative transfer method; (6) use The medium (solution) containing antibiotics was used to ...

Embodiment 3

[0059] Example 3 Cultivation and detection of transgenic engineered cyanobacteria

[0060] This example includes (1) After about 3 weeks of antibiotic screening on the plate, the engineering algae with large algae colonies and dense chlorophyll can be selected, and 1 / 3 of the algae colonies are taken and suspended in a small tube with BG-11 culture medium. Sampling to identify whether it is Anabaena;

[0061] (2) After determining the algae species, take out the algae colony and culture it aseptically in a test tube containing about 2 mL of 25 μg / mL culture solution; after 1 week, transfer it to 30 mL of BG-11 culture solution containing 25-50 μg / mL antibiotic ( 50mL Erlenmeyer flask). After one week, it was expanded to a 250mL Erlenmeyer flask containing 150mL culture solution, and the concentration of antibiotics (kanamycin or thionovin) could be gradually increased from 25μg / mL through 50, 75, 100, 150, 200, 250 increased to 300μg / mL;

[0062] (3) After the antibiotic co...

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Abstract

The invention relates to protonema engineering blue algae with high fat content and high essential fatty acid content and constructed by adopting a reverse vector method. According to the invention, the engineering anabaena is constructed, the expression of a pepc gene contained in the blue algae is regulated and controlled by adopting gene manipulation which includes the fishing of a target gene, the construction of a vector, the transformation of the blue algae, the screening of transformants, the culture and detection of the engineering blue algae and the content measurement of total fat and fatty acid. A result proves that the fat content of the anabaena with the pepc gene reversely expressed is enhanced by 93% compared with that of a wild type, the total fat content is increased from 11.5% of the wild type to 22.2%, and the fat yield is double increased and achieves the mean fat level of eukaryotic algae. Thus, the constructed anabaena with the pepc gene reversely expressed can be taken as a more ideal raw material used for preparing biodiesel and essential fatty acid.

Description

technical field [0001] The invention relates to the fields of biotechnology and microalgae energy, in particular to construct an engineering cyanobacteria to provide raw materials for producing biodiesel and essential fatty acids. Background technique [0002] Which microalgae to use to prepare biodiesel has become one of the keys to the development of bioenergy at home and abroad. In the 35 years since the US Department of Energy launched the "Aquatic Species Program (ASP)" in 1978, scholars from various countries have made a lot of progress in the development of microalgae energy. In the past 6 years, Chisti (2007) and Hu et al. (2008) have further clarified the characteristics of microalgae, which has set off a global upsurge in the production of biodiesel from microalgae. The group of microalgae can be divided into eukaryotic algae and prokaryotic algae. Eukaryotic microalgae have a high lipid content and have always attracted everyone's attention; however, cyanobacter...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/12C12P7/64C12R1/89
CPCY02E50/13Y02E50/10
Inventor 贾晓会施定基候李君米华玲胡文利陈宇清王晓燕田琪琳黄希文何培民
Owner SHANGHAI OCEAN UNIV
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