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Porphyridium chloroplast expression system and application thereof

A chloroplast expression and expression system technology, applied in single-cell algae, introduction of foreign genetic material using vectors, biochemical equipment and methods, etc. problems, to achieve the effect of improving experimental efficiency, low background effect, and low false positive rate

Active Publication Date: 2020-01-10
YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is a lack of tools for genetic transformation, including homologous insertion sites for chloroplast transformation, transformation methods, regulatory sequences, etc., which hinder the development and application of metabolites from P.

Method used

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  • Porphyridium chloroplast expression system and application thereof
  • Porphyridium chloroplast expression system and application thereof
  • Porphyridium chloroplast expression system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Cloning of Endogenous Fragments of Chlorophyllum Chlorophyllum

[0036] According to the published P. chloroplast genome, the following primers were designed and synthesized:

[0037] SEQ1-for: 5'-TATCCGGAATCACTGGGCGTAA-3'

[0038] SEQ1-rev: 5'-TTTAAGGAGGTGATCCAGCCGC-3'

[0039] SEQ2-for: 5'-AAGGGGATATAGCTCAGTTGG-3'

[0040] SEQ2-rev: 5'-GGAGAACCAGCTAGCTCCGGAT-3'

[0041] SEQ3-for: 5'-CATCAACTTTATCTAAAGACGA-3'

[0042] SEQ3-rev: 5'-AATTTTTTTGTTAAATAAAAGTTTTTTGTG-3'

[0043] SEQ4-for: 5'-CTGTATTGTAGTTTTTTTAATA-3'

[0044] SEQ4-rev: 5'-TAATTACTACAATTAGAATTAAACTC-3'

[0045] SEQ5-for: 5'-TCAATAATTAATATTTAGTGTTCA-3'

[0046] SEQ5-rev: 5'-CTGCTATTTTACTTATCACTCATTA-3'

[0047] SEQ6-for: 5'-GATTTATAAAAAACAAAAAAAGCACTTC-3'

[0048] SEQ6-rev: 5'-ACTAGGTGTCCCCTATTATTGGTATG-3'

[0049] Wherein the amplification product of primer SEQ1-for and SEQ1-rev is SEQ ID NO:1; The amplification product of primer SEQ2-for and SEQ2-rev is SEQ ID NO:2; The amplification of pri...

Embodiment 2

[0056] Embodiment 2: Construction of Porphyridium chloroplast expression system

[0057] Design and synthesize the following primers:

[0058] bar-for: 5’-ATGAGCCCAGAACGACGCC-3’

[0059] bar-rev: 5'-TCATCAAATCTCGGTGACGGG-3'

[0060] Using the vector pSVB as a template, PCR amplification was carried out with primers bar-for and bar-rev. The reaction program was: 94°C for 5 min for pre-denaturation; 94°C for 1 min, 54°C for 30 sec, 72°C for 40 sec, a total of 35 cycles ; 72 ° C 5min extension. The PCR amplification product is about 555 bp, which is the herbicide resistance gene bar . After the fragments were subjected to agarose gel electrophoresis, the gel was recovered (Tiangen kit) and purified for later use.

[0061] Based on the above products, pMD18T was used as the starting vector to construct the homologous recombination vector of Porphylococcus chloroplast by homologous recombination method. Among them, pMD-SEQ2, pMD-SEQ3, pMD-SEQ5, and pMD-SEQ6 need to use PCR t...

Embodiment 3

[0079] Example 3 The application of the carrier obtained according to the above examples in the transformation of Porphyrococcus chloroplast

[0080] 1. Construction of Chloroplast Expression Vector of Porphyridium algae

[0081] Design and synthesize the following primers:

[0082] F1-for: 5’- CCTCTAGA ATGCATCATCACCATCATCACCATGGTTTCGGTTGCAACGGTCCCTGG-3'

[0083] F1-rev: 5’-TTAGTAGCACTTGCAGACGAAAATAAATTATCCTTATGAAATGGTGATGGTGATGGTGCAT-3’

[0084] F2-for: 5’-ATGCACCATCACCATCACCATTTCTTCTTCCACATCATCAAGGG-3’

[0085] F2-rev: 5’- GGGATCC TTACTTCCAGACGAGACCGTGGAT-3'

[0086] Among them, F1-for carries Xba I restriction site and 6×His tag, the underlined sequence fragment of F1-rev is SEQ ID NO:7, the underlined sequence in F2-for is 6×His tag, carried by F2-rev Bam HI restriction site.

[0087] Using artificially synthesized antimicrobial peptide gene 1 as a template, PCR amplification was carried out with primers F1-for and F1-rev. The reaction program was: 94°C for 5 ...

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Abstract

The invention relates to the technical field of gene engineering, and particularly relates to a porphyridium chloroplast homologous recombination empty vector and application thereof. The vector comprises an upstream homologous arm, a downstream homologous arm, promoters and terminators, wherein a base sequence of the upstream homologous arm is shown in SEQ ID NO:1 of a porphyridium chloroplast gene group, a base sequence of the downstream homologous arm is shown in SEQ ID NO:2, base sequences of the promoters are shown in SEQ ID NO:3 and SEQ ID NO:4, base sequences of the terminators are shown in SEQ ID NO:5 and SEQ ID NO:6; and a corresponding base sequence which can form a multicistron structure with at least one exogenous gene and is shown in SEQ ID NO:7 is inserted between the corresponding promoter and the corresponding terminator. By adopting the porphyridium chloroplast homologous recombination empty vector disclosed by the invention, stable expression of multiple exogenous genes in the chloroplast can be realized.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a Porphylococcus chloroplast expression system and its application. Background technique [0002] Porphyridium belongs to Rhodophyta, Protoerythrophyta, Porphylococcus, Porphyridaceae, and Porphyridium genus. Porphyridium is a relatively primitive and the only single-celled red algae found so far. Porphyridium is widely distributed and is found in seawater, freshwater, and moist soil. Porphyridium cells are usually spherical, with a diameter of about 5-24 μm. The cells contain a large star-shaped chromophore, rich in phycobiliproteins, of which phycoerythrin has the most content, so the cells appear red or Dark purple. There is usually a layer of viscous sheath outside the Porphyridum cells, that is, Porphyrococcus polysaccharides, and the Porphyrococcus polysaccharides between the cells will adhere to each other and accumulate irregularly. When placed on moist so...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/65C12N1/13C12R1/89
CPCC12N15/65C12N15/8213
Inventor 崔玉琳甄张赫王康任家利秦松
Owner YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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