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Method for transferring exogenous genes into spiral seaweeds with transposons as medium

A technology of exogenous gene and spirulina, applied in the field of genetic engineering, can solve the problems that spirulina has not been reported yet, and achieve the effect of improving efficiency

Inactive Publication Date: 2015-04-01
XIAMEN UNIV
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

Transposon technology has been applied to single-cell cyanobacteria to construct mutant libraries, but it has not been reported in Spirulina

Method used

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  • Method for transferring exogenous genes into spiral seaweeds with transposons as medium
  • Method for transferring exogenous genes into spiral seaweeds with transposons as medium
  • Method for transferring exogenous genes into spiral seaweeds with transposons as medium

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Embodiment Construction

[0043] The present invention will be further described below by embodiment.

[0044] Step 1 Construction of Spirulina transposon-mediated system

[0045] 1.1 Cloning of cyanobacterial transposon sequence (IS)

[0046] Taking the first group as an example, the steps of the other three groups are the same. A set of primers IS1-1, IS1-2 and IS1-3 were designed according to the cyanobacteria IS gene sequence provided on NCBI, wherein Not I, Cla II and EcoR I were respectively set on IS1-1, IS1-2 and IS1-3 site:

[0047] IS1-1:5-ATT GCGGCCGC TTTCGACAACGTTA-3

[0048] Not Ⅰ

[0049] IS1-2:G ATCGAT CCCTGGCGGTTCATAGTG

[0050] Cla Ι

[0051] IS1-3:5-G GAATTC CTAGGCCACTGACT-3

[0052] EcoR I

[0053] In a 0.5ml sterile centrifuge tube, add in sequence: ddH 2 O 37 μl, 10×Taq enzyme buffer 5 μl, 4×dNTP (2.5 mmol / L) 4 μl, IS1-1 primer 1 μl, IS1-3 primer 1 μl, template (Microcystis aeruginosa 6803 genomic DNA) 1 μl, Taq enzyme 1 μl, Total volume 50 μl.

[0054] PCR reaction p...

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Abstract

The invention relates to genetic engineering, in particular to a method for transferring exogenous genes into spiral seaweeds with transposons as a medium. Multiple sets of primers IS1-IS4 are designed according to sequences of known blue-green algae of the NCBI, and transposons elements are cloned from the blue-green algae and implanted into the sequences; ultrasonic transformation and screening of donor plasmids pEGFP-IS-IS-Km are conducted; EcoR I is utilized for digesting pEGFP-IS-IS-Km to obtain linear plasmids, the two ends each have an IS sequence, spiral seaweeds 869s are transformed, and GFP and G418 (npt II) are utilized for screening out mutation-inserted spiral seaweed plants. An integrated spiral seaweed screening system including gfp genes, npt II mark genes and transposons sequences is built, and the gene structure of spiral seaweeds is influenced by random insertion mutation of transposons, so that the quality of spiral seaweeds is changed. The method can be applied to building of a spiral seaweed mutation library, and can also realize introduction and expression of exogenous genes and antisense inhibition regulation of endogenous genes of spiral seaweeds.

Description

technical field [0001] The invention relates to a genetic engineering, in particular to a method for introducing exogenous genes into spirulina by using transposons. Background technique [0002] Spirulina (Spirulina) belongs to Cyanophyta, Oscillatoria, and Spirulina in taxonomy. It is a multicellular, miniature, non-branched, non-helical spiral body that proliferates by division and is a photoautotrophic organism. As a new gene transformation carrier, it has the characteristics of high safety, high nutrition and high nutritional value, and has many unique features. Such as: high tolerance to foreign proteins, rapid reproduction, fast regeneration, simple culture conditions, etc. Spirulina has great potential as a prokaryote with important development and application value. [0003] In recent years, physical and chemical mutagenesis has been widely used to screen target strains. The traditional methods to obtain high-quality Spirulina are generally obtained through natura...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12R1/01
Inventor 章军徐虹丁旭东杜正伟郑天凌
Owner XIAMEN UNIV
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