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Synechocystis efficient double homologous recombinant vector as well as construction method and application thereof

A homologous recombination and construction method technology, applied in the field of genetic engineering, can solve the problems of high-efficiency expression of exogenous genes and few reports, and achieve the effects of increasing content, increasing biomass, and increasing total fatty acid content

Inactive Publication Date: 2013-04-03
山东省农业科学院高新技术研究中心
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the genetic transformation method of Synechocystis sp. PCC6803 is very mature, and there are various methods for constructing its double homologous recombination vector, but there are few reports at home and abroad on the upstream arm of the homologous recombination vector and the high-efficiency expression of foreign genes.

Method used

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  • Synechocystis efficient double homologous recombinant vector as well as construction method and application thereof
  • Synechocystis efficient double homologous recombinant vector as well as construction method and application thereof
  • Synechocystis efficient double homologous recombinant vector as well as construction method and application thereof

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Effect test

Embodiment 1

[0076] Isolation and cloning of cDNA fragments of Synechocystis delta15 fatty acid desaturase gene Delta15 and Delta6 fatty acid desaturase gene Delta6, double homologous recombination fragment psbA2 promoter, psbA2ORF cDNA fragment and Escherichia coli Terminator T1T2 cDNA fragment.

[0077] Cloning of the psbA2 promoter gene fragment of Synechocystis sp. PCC 6803:

[0078] Primers were designed according to the front-end sequence of psbA2ORF in Synechocystis sp. PCC 6803 (accession number: BA000022, AP012205) registered in GenBank, and the first 500 bp of psbA2ORF was used as the promoter sequence (sequence shown in SEQ ID NO: 1, Synchocystis sp. PCC6803), and the design PCR primers:

[0079] Promotor-F: 5'-GATGTCGACGCTTTAGCGTTCCAGTG-3',

[0080] Promotor-R: 5'-CATTTGGTTATAATTCCTT ATGTAT-3',

[0081] The amplification program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, renaturation at 55°C for 30 s, extension at 72°C for 1 min, after...

Embodiment 2

[0109] Construction and transformation of double homologous recombination expression vector in Synechocystis sp. PCC6803

[0110] Through the overexpression technology, the Delta15 gene fragment and the Delta6 gene fragment of Synechocystis sp. PCC6803 were overexpressed in Synechocystis sp. PCC6803. According to the phenotype and fatty acid composition of the transgenic positive Synechocystis sp. Construction method and application in fatty acid synthesis.

[0111] Preparation of plasmid pBluscript SK plus-T1T2:

[0112] Plasmid pKK233-2 (purchased from Clontech Company) for amplifying the E. coli T1T2 terminator, introducing a PstI restriction site at its 5' end and a BamHI restriction site at its 3' end, the primers used are:

[0113] T1T2-F: 5'—ATACTGCAGCCAAGCTTGGCTGTTTTGGC—3';

[0114] T1T2-R: 5’—TTAGGATCCCCCATTATTGAAGCATTTAT—3’;

[0115] The amplification procedure is as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, renaturation at 60°C f...

Embodiment 3

[0123] Detection of Delta15 and Delta6 Gene Expression Levels in Transgenic Synechocystis Positive Plants

[0124] The total RNA was extracted from the transgenic Synechocystis sp. PCC6803 containing the expression vector pSDSy15, the transgenic Synechocystis sp. PCC6803 containing the expression vector pSDSy15Sy6, and the wild type Synechocystis sp. PCC6803 for Real time quantitative PCR analysis. The specific method is as follows:

[0125] Total RNA was extracted from Synechocystis and wild-type Synechocystis with Delta15 and Delta15+Delta6 genes using TRIZOL reagent (purchased from Invitrogen) and liquid nitrogen grinding. The specific operation steps are as follows: take 50ml of cyanobacteria with OD=1.8, centrifuge at 5000rpm at 4°C for 10min to collect the algae cells, grind them in liquid nitrogen to a fine powder, add them to a 1.5ml centrifuge tube, quickly add 1ml TRIZOL (purchased from Invitrogen), Invert and mix well, let stand at room temperature for 5min, centri...

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Abstract

The invention particularly relates to synechocystis efficient double homologous recombinant vector as well as a construction method and the application thereof. The nucleotide sequence of the synechocystis efficient double homologous recombinant vector is shown as SEQ ID NO.8 or SEQ ID NO.9. The invention also discloses the construction method and the application of the synechocystis efficient double homologous recombinant vector. The synechocystis efficient double homologous recombinant vector expresses synechocystis PCC6803Delta 15, Delta 15 and Delta 6 fatty acid desaturase gene in synechocystis PCC6803, and can obviously increase the content of therapic acid in synechocystis.

Description

technical field [0001] The present invention relates specifically to Synechocystis high-efficiency double homologous recombination carrier and its construction method and application, in particular to a high-efficiency double homologous recombination vector for genetic transformation of Synechocystis sp. The application belongs to the technical field of genetic engineering. Background technique [0002] Cyanobacteria, also known as cyanobacteria, are a class of prokaryotic organisms capable of photosynthesis. Cyanobacteria cells can use simple inorganic substances to synthesize organic substances, and the expressed exogenous gene products do not form inclusion bodies, and most cyanobacteria and their extracts are non-toxic to humans and animals, so they are good recipients for transgenic research. Synechocystis sp.strain PCC6803 (Synechocystis sp.strain PCC6803), as a single-cell cyanobacteria, has the characteristics of fast growth, simple culture conditions, no toxin prod...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/66C12N1/13C12P7/64C12R1/89
Inventor 何庆芳陈高毕玉平张燕李伟智杨连群范仲学边斐马德源彭振英
Owner 山东省农业科学院高新技术研究中心
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