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A kind of isochrysis chloroplast homologous recombination transgenic system and its application

A technology of isochrysis and homologous recombination, applied in the field of genetic engineering, can solve the problems of restricting research and application development, lack of chloroplast transformation system, etc., and achieve the effect of improving expression efficiency

Active Publication Date: 2020-04-21
YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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Problems solved by technology

[0005] A high-efficiency chloroplast transgenic system will provide a technical basis for the performance optimization of this algae, but there is little research on genetic engineering of this algae, and there is a lack of chloroplast transformation systems, such as homologous recombination sites that can be stably inserted, and efficient endogenous Regulatory sequences, as well as screening marker genes, etc., which seriously restrict the research and application development of this algae

Method used

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  • A kind of isochrysis chloroplast homologous recombination transgenic system and its application
  • A kind of isochrysis chloroplast homologous recombination transgenic system and its application
  • A kind of isochrysis chloroplast homologous recombination transgenic system and its application

Examples

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Effect test

Embodiment 1

[0039] Example 1 Cloning of Endogenous Fragments of Isochrysis Chlorophyllum

[0040] According to the published Isochrysis chloroplast genome, the following primers were designed and synthesized:

[0041] SEQ1-for: 5'-ATGCGTAGATATTAGAAAGAACACCGG-3'

[0042] SEQ1-rev: 5'-TGGTGGGCCATCCAGGACTTGAACC-3'

[0043] SEQ2-for: 5'-GGGGGTATAGCTCAGTTGGTAGAGC-3'

[0044] SEQ2-rev: 5'-ATCCCCCAATGTCTCACTAACTAAC-3'

[0045] SEQ3-for: 5'-ATAAATATAATTTAGCTAATAAA-3'

[0046] SEQ3-rev: 5'-GATTATATTGTTTTGTTTAAA-3'

[0047] SEQ4-for: 5'-TTACTTTTTTTGTACTCAATTATAAAAT-3'

[0048] SEQ4-rev: 5'-GACAACCTCCTTGGTTTAAGATGTTATGC-3'

[0049] SEQ5-for: 5'-TTACTAGTATCCGACATACCGACT-3'

[0050] SEQ5-rev: 5'-CTTTTTAATTAGATTTTTATTAATAAAGTA-3'

[0051]SEQ6-for: 5'-TTAGGTCAAGATTTTATATCTAGC-3'

[0052] SEQ6-rev: 5'-CTGAAAAATTGTGTACAATACTTTTTTCAG-3'

[0053] 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:...

Embodiment 2

[0060] Embodiment 2: Construction of Isochrysis chloroplast expression system

[0061] Design and synthesize the following primers:

[0062] bar-for: 5’-ATGAGCCCAGAACGACGCC-3’

[0063] bar-rev: 5'-TCATCAAATCTCGGTGACGGG-3'

[0064] Using the commercial carrier 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 Cycling; 72 °C 5 min 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.

[0065] Based on the above products, pMD18T was used as the starting vector to construct the Isochrysis chloroplast homologous recombination vector by homologous recombination method. Among them, pMD-SEQ2, pMD-SEQ3, pMD-SEQ5, and pMD-SEQ6 need to...

Embodiment 3

[0083] Example 3 Application of the Vectors Obtained According to the Examples Above in the Transformation of Isochrysis Chloroplasts

[0084] Next, two antimicrobial peptide genes (GenBank No.6K50_A; GenBank No.AKA60777.2) with antibacterial activity were inserted into the empty vector (pTl / ch / bar) obtained above and then introduced into Isochrysis. The expression of the source gene was used to test the performance of the vector.

[0085] 1. Construction of expression vector

[0086] Design and synthesize the following primers:

[0087] F1-for: 5’- CCTCTAGA ATG CATCATCACCATCACCAT GGTTTCGGTTGCAACGGTCCCTGG-3'

[0088] F1-rev: 5’-TTAGTAGCACTTGCAGACGAA GAAACTAAG GAAATGGTGATGGTGATGGTGCAT-3'

[0089] F2-for: 5’-ATG CACCATCACCATCACCAT TTCTTCTTCCACATCATCAAGGG-3'

[0090] F2-rev: 5’- GGGATCC TTACTTCCAGACGAGACCGTGGAT-3'

[0091] 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 seq...

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Abstract

The invention relates to the technical field of biological genetic engineering, in particular to a method for constructing an isochrysis sp. chloroplast homologous recombinant transgenic system. Sequences shown as SEQ ID NO: 1 and SEQ ID NO: 2 derived from an isochrysis sp. chloroplast genome are taken as homologous arms; sequences shown as SEQ ID NO: 3 and SEQ ID NO: 4 are taken as promoters; sequences shown as SEQ ID NO: 5 and SEQ ID NO: 6 are taken as terminators, a bar gene is taken as a screening marker gene, a plurality of exogenous genes are connected through SEQ ID NO: 7 to form a polycistron structure so as to construct a homologous recombinant vector, the vector is introduced into isochrysis sp. cells by using a gene gun method, and a transgenic algal strain is obtained through herbicide glufosinate-ammonium screening. By adopting the isochrysis sp. chloroplast homologous recombinant transgenic system, a plurality of exogenous genes can be stably expressed in chloroplast.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an Isochrysis chloroplast homologous recombination transgene system and its application. Background technique [0002] Chloroplast is an important organelle in higher plant cells and eukaryotic microalgal cells capable of photosynthesis. It contains a complete set of relatively independent genetic material. The source of chloroplasts is generally considered to be prokaryotic cells endocytosed by eukaryotic cells. Its genetic material is prokaryotic in nature. Its characteristics include: no intron structure inside the gene, multiple genes can be fused in one polycistronic joint expression, etc. According to the characteristics of chloroplast genome, chloroplast genetic transformation technology has been developed rapidly. Compared with the nuclear transformation technology, the chloroplast transformation technology has obvious advantages: homologous recombination, no...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12R1/89
CPCC12N15/8213C12N15/8214
Inventor 崔玉琳王寅初王康任家利焦绪栋李莉莉秦松
Owner YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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