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Method for constructing Platymonas subcordiformis chloroplast expression system

A chloroplast expression and subcardioid technology, applied in the field of biological genetic engineering, can solve the problem of limited genetic transformation of subcardioid flat algae, and achieve the effects of stable expression, high expression and high expression efficiency.

Inactive Publication Date: 2014-01-08
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
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Problems solved by technology

But so far, the research on the genetic transformation of P. subcardiac is limited to nuclear genome manipulation, and the field of chloroplast genome manipulation is still completely blank.

Method used

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  • Method for constructing Platymonas subcordiformis chloroplast expression system
  • Method for constructing Platymonas subcordiformis chloroplast expression system
  • Method for constructing Platymonas subcordiformis chloroplast expression system

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Embodiment

[0032] Example: Achieve stable expression of herbicide resistance gene in Platymonas subcordiformis chloroplast

[0033] 1. Amplification and cloning of two chloroplast homologous recombination fragments of Platymonas subcordiformis

[0034] Design and synthesize the following two pairs of primers

[0035] P1: 5’-CGG GGTACC TCCTACGGGAGGCAGCAGT-3' (underline is Kpn I restriction site)

[0036] P2: 5’-GC GTCGAC TTGAGGCAAATGGGCTATGC-3' (underline is Sal I restriction site)

[0037] P3: 5’-TT GCGGCCGC ACAACGGAGTTTCGGAATA-3' (underline is Not I restriction site)

[0038] P4: 5’-C GAGCTC CGTTACTCAAACCGACATTC-3' (Sac I restriction site is underlined)

[0039] The amplification product of primers P1 and P2 is SEQ ID NO: 1, which is the fragment 16S-trnI; the amplification product of primers P3 and P4 is SEQ ID NO: 2, which is the fragment trnA-23S (see figure 1 ).

[0040] Using the total DNA of Platymonas subcordiformis as a template, PCR amplification was carried out with primers P1 and P2. ...

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Abstract

The invention relates to the biological gene engineering technical field, concretely relates to a method for constructing a Platymonas subcordiformis chloroplast expression system. According to the invention, a sequence shown in SEQ ID NO:1, a sequence shown in SEQ ID NO:2 and a DNA recombinant fragment constructed by exogenous gene with selective label are used to insert a cloning vector and introduced into Platymonas subcordiformis cell, conversed and cultured and screened to obtain the transgene Platymonas subcordiformis. The stable expression system of the Platymonas subcordiformis chloroplast can realize the stabilization and highly expression of exogenous gene in the Platymonas subcordiformis chloroplast, and greatly accumulate the target protein protein, and has significance for improving the algae variety and exploiting the gene engineering products.

Description

Technical field [0001] The present invention relates to the technical field of biological genetic engineering, in particular to a method for constructing a chloroplast expression system of subcordiform flat algae. Background technique [0002] In eukaryotic cells capable of photosynthesis, the three organelles of nucleus, chloroplast and mitochondria all contain DNA molecules, which constitute an independent and interconnected genetic system. Since the birth of genetic engineering technology in the 1970s, the transformation technology of introducing foreign genes into the nucleus has been widely used. However, with the in-depth development of research, people gradually realized that genetic engineering operations in the nuclear genome have the following insurmountable difficulties: 1. The nuclear genome is huge and the background is complex; 2. The imported foreign genes are randomly inserted into the nuclear genome; 3. . The expression efficiency of foreign genes is low and the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63
Inventor 崔玉琳姜鹏陈华新李富超秦松
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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