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Novel promoter for use in transformation of algae

A genetic transformation method and promoter technology, which are applied in the direction of introducing foreign genetic material, unicellular algae, biochemical equipment and methods using vectors, etc., can solve the problems such as no reports of successful genetic transformation of Xenorhabdus spp., and achieve high photosynthesis. effect of ability

Inactive Publication Date: 2014-05-07
NAT UNIV CORP KOCHI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, according to other documents (non-patent document 4), no matter how hard they try, there is no report on the success of genetic transformation of Gynostomia sp.

Method used

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  • Novel promoter for use in transformation of algae
  • Novel promoter for use in transformation of algae
  • Novel promoter for use in transformation of algae

Examples

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preparation example Construction

[0049] As a preparation method of the carrier of the present invention, a usual method can be used. For example, each of the above-mentioned sequences and the donor vector may be cleaved with a restriction endonuclease, annealed, and then ligated with a DNA ligase. Alternatively, each sequence can also be cloned into a vector by a simple known method using a clonase reaction.

[0050] The method for genetic transformation of algae according to the present invention is characterized by comprising the steps of preparing the vector and introducing the vector into algae cells.

[0051] The preparation method of the carrier of the present invention is as described above, and methods known to those skilled in the art can be used.

[0052] As a method for introducing the vector of the present invention into algae cells, known methods such as gene gun method, glass bead stirring method, microinjection method, Agrobacterium method (Agrobacterium method), lithium acetate method, calciu...

Embodiment 1

[0057] The separation of the ClorDNA virus promoter that the present invention relates to

[0058] (1) Extraction of genomic DNA of viruses that infect marine algae

[0059] According to Tomaru, Y. et al., Aquatic Microbial Ecology, vol.50, pp.103-112 (2008) method recorded, from the central order diatom chaetoceros cf. lorenzianus) DNA virus (ClorDNA virus) genome extraction.

[0060] Specifically, first, the virus liquid (10 mL) was filtered through a 0.22 μm filter (Millex-GS, manufactured by MILLIPORE, pore size: 0.22 μm) to remove fragments of algae cells and the like. A 40% polyethylene glycol 6000 solution (manufactured by Woko) was added to the obtained filtrate so that the final concentration became 10 w / v%, and it was left to stand at 4° C. overnight. The solution was transferred to a centrifuge tube (Nalgen, UltraBottle Assemblies), centrifuged at 57,000×g, 4°C for 1.5 hours using an ultracentrifuge (Beckman, Ultracentrifuge L8-70M), and the supernatant was remove...

Embodiment 2

[0066] Preparation of the carrier containing the ClorDNA virus promoter involved in the present invention

[0067] (1) Preparation of each imported clone plasmid

[0068] Using the Multisite Gateway (registered trademark) Pro Kit (manufactured by Invitrogen Co., Ltd.), the ClorDNA virus promoter obtained in the above-mentioned Example 1 and the nourthricin-resistant gene (nat) (Poulsen , N. et al., Jornal of Phycology, 42, pp1059-1065.(2006)), and the fcp terminator of Thlassiosira pseudonana (Poulsen, N. et al., Jornal of Phycology, 42, pp 1059- 1065. (2006)) import cloning plasmid.

[0069]Specifically, using the High pure PCR Cleanup Micro Kit (manufactured by Roche), according to its instruction manual, the ClorDNA virus promoter solution obtained in the above Example 1 having the attB sequence necessary for the clonase reaction for constructing the plasmid was purified. Refined. Next, this solution (50 fmoles) was mixed with a donor vehicle (manufactured by Invitrogen,...

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Abstract

The problem to be solved by the present invention is to provide a highly-efficient transformation technology, specifically, a highly-efficient promoter used for transforming algae, a vector comprising the promoter, and a method for transforming algae by using the vector. The promoter according to the present invention is characterized in comprising a polynucleotide constituting a non-coding region located upstream of a gene encoding a structural protein of a ClorDNA virus, and the like.

Description

technical field [0001] The present invention relates to a novel promoter for genetic transformation of algae, a vector containing the promoter, and a method for genetic transformation of algae using the vector. Background technique [0002] Utilizing sunlight, which is a constant and stable energy source, is very important in terms of energy countermeasures. The photosynthesis of plants is a good system that can most effectively convert sunlight energy into chemical energy. It not only absorbs and assimilates carbon dioxide or excess nutrients in the environment, but also releases oxygen. Therefore, technology development using plants is expected to be a technology that can solve energy problems. [0003] Among plants, since algae live in seawater and fresh water that exist in large quantities, the amount of algae is also quite large. In addition, algae have a remarkable photosynthetic ability. In addition, among algae, there are species that produce useful compounds such ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N1/13
CPCC12N15/8216C12N15/79
Inventor 足立真佐雄冈见卓马长崎庆三外丸裕司
Owner NAT UNIV CORP KOCHI UNIV
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