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Method for importing exogenous DNA (deoxyribonucleic acid) into nannochloropsis oculata chloroplast and related chloroplast genome sequence

A chloroplast genome, Nannochloropsis technology, applied in the field of biological genetic engineering, to achieve the effect of stable expression

Active Publication Date: 2014-06-04
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method has not been used for genetic modification of chloroplasts in single-celled microalgae
However, there are no reports on the genome sequence of Nannochloropsis chloroplasts and the genetic transformation of Nannochloropsis chloroplasts.

Method used

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  • Method for importing exogenous DNA (deoxyribonucleic acid) into nannochloropsis oculata chloroplast and related chloroplast genome sequence
  • Method for importing exogenous DNA (deoxyribonucleic acid) into nannochloropsis oculata chloroplast and related chloroplast genome sequence
  • Method for importing exogenous DNA (deoxyribonucleic acid) into nannochloropsis oculata chloroplast and related chloroplast genome sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0057] Embodiment: Realize the stable expression of green fluorescent protein GFP gene in Nannochloropsis chloroplast

[0058] 1. Validation of the restriction modification system of Nannochloropsis

[0059] The specific restriction endonucleases in bacterial cells are used to resist the invasion of phages, and this system is called the restriction modification system. This system is also present in cyanobacteria. The evolutionary status of unicellular eukaryotic microalgae is unique, whether there is a similar restriction modification system. To do this, use the following steps to verify:

[0060](1) Extraction of total protein from Nannochloropsis: Cultivate Nannochloropsis to logarithmic phase, 5000g, 10min, collect algae at 4°C; 5ml PENA solution (10mM K3PO4, 10mM EDTA, 50mM NaCland a tablet of aprotinin per 50ml, pH 7.0) rinse twice; suspend the algae in 1ml PENA solution; add 100mg glass beads, vortex vigorously for 1min; centrifuge at 4000g, 15min, 4°C; take the supe...

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Abstract

The invention relates to the technical field of biological gene engineering, and particularly relates to a method for importing exogenous DNA (deoxyribonucleic acid) into nannochloropsis oculata chloroplast and a related chloroplast genome sequence. The gene sequence comprises a sequence shown by SEQ ID No.1, a sequence shown by SEQ ID No.2 and an exogenous gene inserted between the two sequences. The method comprises the concrete step of importing a chloroplast transformation vector comprising the sequence shown by SEQ ID No.1, the sequence shown by SEQ ID No.2 and the exogenous gene inserted between the two sequences into nannochloropsis oculata cells by an electroporation process. The method provided by the invention can be used for designing and establishing a chloroplast genetic modification system universal in nannochloropsis oculata and a chloroplast genetic modification system universal in multiple species of unicellular microalgae according to the conserved genes among the genomes of seven nannochloropsis oculata chloroplasts and the conserved genes of the nannochloropsis oculata chloroplast and diatom or brown algae chloroplast genome.

Description

technical field [0001] The invention relates to the technical field of biogenetic engineering, in particular to a method for introducing exogenous DNA into Nannochloropsis chloroplasts and related chloroplast genome sequences. technical background [0002] The shortage of energy and resources has become a major strategic issue that restricts the sustainable development of my country's economy and affects my country's national security. Based on the characteristics of clean, efficient, and renewable biocatalysts, biomanufacturing can greatly reduce the dependence of industrial development on fossil resources, reduce energy consumption, material consumption, water consumption, and waste discharge, significantly improve economic benefits, and enhance market competitiveness. Compared with petrochemical routes, bio-manufacturing can save energy by an average of 30-50%, and reduce human impact on the environment by 20-60%. Sunlight is the most abundant clean energy on earth. Pho...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/79C12R1/89
Inventor 路延笃徐健辛一魏力
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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