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Rich-lipid nannochloropsis chloroplast transgenic system and application thereof

A technology of Nannochloropsis and chloroplasts, which is applied in the field of genetic engineering, can solve the problems that hinder the progress of basic research and application development, that there is no technology for transforming the chloroplasts of Lipid-rich Nannochloropsis, and affect the expansion of mutant strains, etc. Effects of photosynthetic growth rate, highly efficient endogenous regulatory sequences, and stable insertion

Active Publication Date: 2020-02-04
YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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AI Technical Summary

Problems solved by technology

[0006] The chloroplast genome of Lipid-rich Nannochloropsis has been sequenced, which provides data support for the chloroplast transformation technology; at the same time, the nuclear transformation technology of the related species of Nannochloropsis also provides a basis for the chloroplast transformation method of Lipid-rich Nannochloropsis. However, the light-independent protochlorophyllide reductase (chlL) gene is selected as the homologous insertion site for the chloroplast transformation of Nannochloropsis, and the mutation of this gene will affect the expansion of the mutant strain
So far, there is no research on the transformation technology of lipid-rich Nannochloropsis chloroplasts, especially the endogenous homologous recombination sites and regulatory sequences that can achieve stable inheritance, which hinders the progress of basic research and application development of this algae

Method used

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  • Rich-lipid nannochloropsis chloroplast transgenic system and application thereof
  • Rich-lipid nannochloropsis chloroplast transgenic system and application thereof
  • Rich-lipid nannochloropsis chloroplast transgenic system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Cloning of Endogenous Fragments of Lipid-rich Nannochloropsis Chlorophyllum

[0037] Design and synthesize the following primers:

[0038] SEQ1-for: 5'-CTAGGAGACTTGAGTATAGTAGG-3'

[0039] SEQ1-rev: 5'-CTGGGCTATCCTGGACTTGAACCA-3'

[0040] SEQ2-for: 5'-TGGGGGTATAGCTCAGCTGGTAGAG-3'

[0041] SEQ2-rev: 5'-TCGGGGAGAACCAGCTAGCTCCGG-3'

[0042] SEQ3-for: 5'-ATTTTTGTTGTTGACTAAAACAC-3'

[0043] SEQ3-rev: 5'-AATGAGTACTAGAATAAATAGGG-3'

[0044] SEQ4-for: 5'-GAACATAAAAGAGGTTTTAAAATTACT-3'

[0045] SEQ4-rev: 5'-TATAGCAGTAAATTCTCTGGTTCTCG-3'

[0046] SEQ5-for: 5'-ATAATACATAACAACTAAAACCAA-3'

[0047]SEQ5-rev: 5'-GTACAATGTGTTAGGTCTTACAAAT-3'

[0048] SEQ6-for: 5'-GAATATTTAATTACATATGAGTGA-3'

[0049] SEQ6-rev: 5'-ATTAAATTAACAAAGAAAATCTG-3'

[0050] 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 primer SEQ3-for and SEQ3-rev Product is SEQ I...

Embodiment 2

[0057] Example 2: Construction of an empty vector for lipid-rich Nannochloropsis chloroplast transformation

[0058] Design and synthesize the following primers:

[0059] bar-for: 5’-ATGAGCCCAGAACGACGCC-3’

[0060] bar-rev: 5'-TCATCAAATCTCGGTGACGGG-3'

[0061] Using the vector pSVB as a template, carry out PCR amplification with primers bar-for and bar-rev. The reaction program is: 94°C 5 min pre-denaturation; 94°C 1 min, 54°C 30 sec, 72°C 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 fragment was subjected to agarose gel electrophoresis, the PCR product purified by gel recovery (Tiangen company kit) was connected to the pMD-18T vector (Sigma company) to obtain a gene containing bar The recombinant plasmid pMD- bar .

[0062] Based on the above products, pMD18T was used as a starting vector to construct a lipid-rich Nannochloropsis chloroplast homologous recombi...

Embodiment 3

[0079] Example 3 Application of the carrier obtained according to the above examples in the transformation of lipid-rich Nannochloropsis chloroplast

[0080] Next, two antimicrobial peptide (GenBank No.6K50_A; GenBank No. AKA60777.2) genes with antibacterial activity were inserted into the vector and then introduced into Nannochloropsis lipophilicum. The expression of these two foreign genes was detected to detect the carrier performance.

[0081] 1. Construction of expression vector

[0082] Design and synthesize the following primers:

[0083] F1-for: 5’- CCTCTAGA ATG CATCATCACCATCACCAT GGTTTCGGTTGCAACGGTCCCTGG-3'

[0084] F1-rev:

[0085] TGGTGATGGTGATGGTGCAT AAAATATCTACTC TTAGTAGCACTTGCAGACGAA

[0086] F2-for: 5’-ATG CACCATCACCATCACCAT TTCTTCTTCCACATCATCAAGGG-3'

[0087] F2-rev: 5’- GGGATCC TTACTTCCAGACGAGACCGTGGAT-3'

[0088] 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...

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Abstract

The invention relates to the technical field of genetic engineering, in particular to a rich-lipid nannochloropsis chloroplast transgenic system and application thereof. A carrier of the system comprises at least one promoter, a terminator, an upstream homologous arm and a downstream homologous arm, wherein the upstream homologous arm has a base sequence shown as SEQ ID NO:1; the downstream homologous arm has a base sequence shown as SEQ ID NO:2; and a base sequence, shown as SEQ ID NO:7, forming a polycistron structure with at least one exogenous gene is inserted between the homologous arms.By using the rich-lipid nannochloropsis chloroplast transgenic system provided by the invention, the stable expression of a plurality of exogenous genes in chloroplasts can be realized.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a lipid-rich Nannochloropsis chloroplast transgenic system and its application. Background technique [0002] In eukaryotic cells capable of photosynthesis, there are three sets of relatively independent genetic material, namely, nuclear chromosomes, chloroplast genomes and mitochondrial genomes. The nuclear chromosomal DNA is the genetic material of eukaryotic nature, which encodes the genetic information necessary for cell survival. Chloroplast and mitochondrial genomes are mostly prokaryotic in nature and encode genetic information related to organelle function. From an evolutionary perspective, chloroplasts and mitochondria are believed to be derived from prokaryotic cells such as eukaryotic cells endocytic bacteria. Most of the chloroplasts and mitochondria rely on the function of the chromosomal genome in the nucleus, and the key proteins of some organelle fun...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/65C12R1/89
CPCC12N15/65C12N15/8213C12N2830/20
Inventor 崔玉琳王康任家利王寅初秦松
Owner YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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