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DNA sequence and application thereof

A DNA sequence and nucleotide sequence technology, applied in the field of genetic engineering, can solve problems such as cell death, unfavorable industrial application, and tediousness

Active Publication Date: 2017-06-13
JINAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the effect of modifying a single key gene in the fatty acid synthesis pathway of microalgae is also limited.
In response to this problem, some researchers overexpressed multiple genes at the same time to obtain higher oil production traits, but this operation process is very time-consuming and cumbersome
[0004] At the same time, one of the factors restricting the development of microalgae-based biodiesel is that the cell wall of microalgae is very thick, and the extraction of intracellular oil needs to remove the cell wall of microalgae, which will increase the production cost and cause cell death, which is very important for industrial It is very disadvantageous for the application
Although some non-cell destruction oil extraction methods have been successful, they still cannot meet the needs of industrial oil production from microalgae

Method used

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  • DNA sequence and application thereof
  • DNA sequence and application thereof
  • DNA sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: Construction of BZIP transcription factor overexpression vector

[0043]BZIP transcription factor of the present invention contains 856 amino acids, uses SMART website and NCBI website to analyze the protein sequence of putative BZIP transcription factor, finds that its BZIP transcription factor contains 2 structural domains: FBPcase structural domain (12-399), BZIP Domain (499-543) ( figure 1 A).

[0044] Genomic DNA of Nannochloropsis was extracted (Meiji DNA Extraction Kit). Primers (Shanghai Sangon Bioengineering Co., Ltd.) were purified by PAGE and diluted to 20 μM when used. The primer pair Na07F, Na08R (Table 1) was used to amplify the sequence of the BZIP transcription factor with the genome of Nannochloropsis as a template, the full length of which is 4057bp.

[0045] Table 1. Related primers

[0046]

[0047] Table 2. PCR reaction system (20μl)

[0048]

[0049] The reaction conditions are: pre-denaturation: 94°C, 2min.

[0050]

...

Embodiment 2

[0054] Embodiment 2: Transformation of Nannochloropsis and screening of transformed algae

[0055] In the first step, the recombinant overexpression vector (pna03-[BZIP]TF) was linearized using ScaI restriction endonuclease (TAKARA), incubated for 8h, and then purified using a DNA fragment purification kit (takara); the second step , Transform the linearized recombinant overexpression vector by electric shock, the method is as follows: 4400rpm, 10min centrifugation to collect algae cells, use 375mmol sterile sorbitol to wash 3 times, remove the seawater inside as much as possible, finally, use 150μl sterile sorbitol The solution was used to resuspend the algal cells, add 30ug of pre-cooled silicon essence DNA (heated at 95 degrees for 1min), and 3ug of linearized recombinant overexpression vector, let it stand on ice for 10min, and use an electric shock device for electric shock, the conditions are as follows: 2.2 kv, the capacitance is 50Uf, and the capacitance is 600ohms. I...

Embodiment 3

[0056] Example 3: Verification of Transformed Algae

[0057] The first step is to verify at the molecular level, centrifuge 100ml of transformed algae (BZIP-TF1 and BZIP-TF2) and 100ml of wild-type Nannochloropsis at 4400rpm for 10min, collect the microalgae, and extract genomic DNA (Meiji DNA Extraction Kit ), utilize primer Na01 and Na02 to carry out PCR, PCR condition, system refer to embodiment 1, the electrophoresis band of 4200bp has occurred in transformed algae (such as figure 2 shown), but not in the control algae; in the second step, the electrophoretic bands were recovered by cutting the gel, sequenced with primer Na01, and blasted on the NCBI network. image 3 The results showed that the 4057bp electrophoresis band was indeed the gene sequence of BZIP transcription factor. It shows that the recombinant overexpression vector (pna03-[BZIP]TF) is successfully integrated into the genome of microalgae.

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Abstract

The invention relates to the field of gene engineering, and in particular relates to a DNA sequence and application thereof. The DNA sequence encodes a BZIP transcription factor, the BZIP transcription factor comprises a FBPcase structure domain and a BZIP structure domain, and the BZIP transcription factor can be used for up-regulation of a plurality of key genes in a TAG (triacylglycerol) and fatty acid synthesis pathway and down-regulation of cell wall synthesis related genes, so that the DNA sequence can be used in the field of microalgae-type bio-oil production, and has a good application prospect.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a DNA sequence and its application. Background technique [0002] Many species of microalgae are rich in nutrients such as protein, lipid and various vitamins, and have been widely used in nutrition and health care, bait, etc. The oil content and fatty acid composition of different types of microalgae are quite different, and microalgae are regarded as an important source of large-scale development of bio-oil and polyunsaturated fatty acid products such as EPA and DHA. In addition, lipid-rich microalgae are also considered to be promising new raw materials for biodiesel production. Petroleum resources are depleting day by day, and there is an urgent need to develop clean and renewable energy sources. Biodiesel produced by microalgae has many advantages such as fast growth and high oil production, and has attracted people's attention. [0003] Previous studies have found that ...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/79C12N1/13C12P7/64C12R1/89
CPCC07K14/405C12N15/79C12P7/6409C12P7/6463C12P7/649Y02E50/10
Inventor 李宏业李达伟杨维东刘洁生
Owner JINAN UNIVERSITY
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