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Recombinant vector over-expressing glucose-6-phosphate dehydrogenase gene, and its application

A glucose phosphate and recombinant carrier technology, which is applied in the direction of recombinant DNA technology, microorganism-based methods, and the use of vectors to introduce foreign genetic materials, etc., can solve the problems of lack of research on G6PD, and achieve convenient cloning operations, high startup efficiency, and suitable for transformation. Effect

Inactive Publication Date: 2014-11-05
JINAN UNIVERSITY
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Problems solved by technology

However, so far G6PD has been lack of research in plants and algae

Method used

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  • Recombinant vector over-expressing glucose-6-phosphate dehydrogenase gene, and its application
  • Recombinant vector over-expressing glucose-6-phosphate dehydrogenase gene, and its application
  • Recombinant vector over-expressing glucose-6-phosphate dehydrogenase gene, and its application

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Experimental program
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Effect test

Embodiment 1

[0054] 1. Construction of expression vector pHY18

[0055] 1. Analysis, cloning and connection of expression vector-related elements

[0056] The recombinant vector for overexpressing the glucose-6-phosphate dehydrogenase gene of the present invention is obtained by transforming the commercialized vector pMD19-T (Takara, Dalian, China), and mainly has two expression cassettes, the resistance screening gene CAT expression cassette and target gene expression cassettes.

[0057] The original expression control sequence was obtained by using NEB high-fidelity Pfu polymerase PCR and overlapping PCR (for specific PCR system and procedures, refer to Bryksin and Matsumura, 2010). The primers for the expression control sequence elements are: NR promoter (Pt13, Pt14), NR terminator (Pt79, Pt80). The CAT expression cassette was cloned from plasmid pHY11 using primers Pt83 and Pt84 (see Niu et al., 2012 for details). The PNR-PP3-SD fragment was amplified with primers Pt13 and Pt82. Th...

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Abstract

The invention discloses a recombinant vector over-expressing a glucose-6-phosphate dehydrogenase gene, and its application. The recombinant vector comprises a screening labeled gene expression cassette and a target gene expression cassette; the screening labeled gene expression cassette comprises a resistance screening gene, the upstream of the resistance screening gene is fused with an Escherichia coli P1 promoter, and the downstream of the resistance screening gene is fused with an Escherichia coli ccdB terminator; and the target gene expression cassette comprises a Phaeodactylum tricornutum fcpC gene promoter, the glucose-6-phosphate dehydrogenase gene, a Phaeodactylum tricornutum fcpA gene terminator and an Omega leader sequence, and the Omega leader sequence is in the downstream of the Phaeodactylum tricornutum fcpC gene promoter. The recombinant vector is small, is suitable for converting, is easy to operate, is a homologous sequence of the Phaeodactylum tricornutum, and benefits for expression. The over-expression of the glucose-6-phosphate dehydrogenase gene can substantially improve the content of lipids in micro-algal cells, and lays the foundation for the researches of biological energy.

Description

Technical field: [0001] The invention belongs to the field of microalgae genetic engineering, and in particular relates to a recombinant vector for overexpressing a 6-phosphate glucose dehydrogenase gene and its application. Background technique: [0002] The non-renewability of traditional energy determines the trend of its depletion and causes increasingly serious environmental problems. The increasingly serious energy crisis and environmental pollution have aroused people's awareness of energy worries and the concept of zero pollutant discharge, strengthened the recycling and scientific utilization of waste oil and industrial waste, and promoted the development and utilization of clean new energy. Seeking a safe, reliable and clean renewable energy has become the focus of common attention in the world today. Bioenergy refers to the energy produced by using biorenewable raw materials and solar energy, including biomass energy, bioliquid fuel and energy produced by using b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C12N1/13C12R1/89
Inventor 李宏业薛姣杨维东刘洁生
Owner JINAN UNIVERSITY
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