A kind of lipid-rich Nannochloropsis chloroplast transgenic system and its application

A technology of Nannochloropsis and chloroplasts, which is applied in the field of genetic engineering, can solve the problems affecting the expansion of mutant strains, hindering the progress of basic research and application development, and the lack of lipid-rich Nannochloropsis chloroplast transformation technology. Effects of photosynthetic growth rate, highly efficient endogenous regulatory sequences, and stable insertion

Active Publication Date: 2020-05-01
YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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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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  • A kind of lipid-rich Nannochloropsis chloroplast transgenic system and its application
  • A kind of lipid-rich Nannochloropsis chloroplast transgenic system and its application
  • A kind of lipid-rich Nannochloropsis chloroplast transgenic system and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Cloning of endogenous fragments of chloroplasts

[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] The amplification product of primers SEQ1-for and SEQ1-rev is SEQ ID NO:1; the amplification product of primers SEQ2-for and SEQ2-rev is SEQ ID NO: 2; the amplification product of primers SEQ3-for and SEQ3-rev The product is SEQ ID NO: 3; the amplification product of primers SE...

Embodiment 2

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

[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, PCR amplification was carried out with primers bar-for and bar-rev. The reaction program was: 94℃ 5min pre-denaturation; 94℃ 1 min, 54℃ 30 sec, 72℃ 40 sec, a total of 35 cycles; Extension at 72°C for 5 min. The PCR amplification product is about 555 bp, which is the herbicide resistance gene bar . After the fragment was electrophoresed on agarose gel, the PCR product purified by gel recovery (Tiangen kit) was connected with pMD-18T vector (Sigma) to obtain the gene bar Recombinant plasmid pMD- bar .

[0062] Based on the above products, pMD18T was used as the starting vector to construct a homologous recombination vector for the chloroplast of Pseudochlorococcum lipid-rich microalgae thro...

Embodiment 3

[0079] Example 3 Application of the vector obtained according to the above example in the transformation of chloroplasts of Pseudochlorococcum lipo-rich

[0080] Next, insert the two antibacterial peptides (GenBank No.6K50_A; GenBank No.AKA60777.2) genes with antibacterial activity into the vector and then introduce them into the microsphaeropsis lipid-rich, and detect the expression of these two foreign genes. The performance of the carrier.

[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 SEQID NO: 7, the underlined sequence i...

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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 chloroplast transgene system of Pseudochlorococcum lipid-rich and its application. Background technique [0002] In eukaryotic cells capable of photosynthesis, they contain three sets of relatively independent genetic material, namely nuclear chromosomes, chloroplast genome and mitochondrial genome. The chromosomal DNA of the cell nucleus is the genetic material of eukaryotic nature, which encodes the genetic information necessary for cell survival. Chloroplast and mitochondrial genomes are mostly prokaryotic and encode genetic information related to organelle functions. From an evolutionary perspective, chloroplasts and mitochondria are derived from eukaryotic cells endocytosing bacteria and other prokaryotic cells. Chloroplasts and mitochondria mostly rely on the functions of the chromosomal genome in the nucleus. Some of the key proteins for organelle functions are ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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