Method for screening spiral seaweeds through gene transformation

Abstract

The invention provides a method for screening spiral seaweeds through gene transformation, and relates to genetic engineering. The method comprises the steps that (1) a spiral seaweed integration and screening plasmid pEGFP-IS-IS-Km containing two inserting sequences, a gfp gene, a marker gene npt II and a marker gene Ap is established, (2) ultrasonic conversion and screening are conducted on a donor plasmid pEGFP-IS-IS-Km, wherein enzyme digestion is conducted on the pEGFP-IS-IS-Km through EcoRI, a linear plasmid is obtained, the two ends of the linear plasmid are each provided with an IS sequence, the linear plasmid is similar to the characteristic structure of a transposon, the spiral seaweed 869s is converted through a laboratory ultrasonic conversion method, benzylpenicillin and G418 (nptII) are used for selecting out spiral seaweed strains with insertion mutations, mutant strains are selected out through a two-step screening method, a resistant plate is used for primary screening, after gradient dilution is completed, a fluorescent microscope is used for picking out singular green fluorescent seaweeds, then ultrasonic secondary screening is conducted, enlarge cultivation is carried out, and seaweed strains with excellent performance are selected out.

Description

technical field [0001] The invention relates to a genetic engineering, in particular to a method for transforming and screening spirulina genes. 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, and cultivation The conditions are simple and so on. 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...

AI Technical Summary

Problems solved by technology

The screening marker system commonly used in the above experiments is to use antibiotic genes as screening markers, but there are different opinions on what kind of antibiotics to choose and the concentration of antibiotics. Hybridization signals are often unstable, and even traits degenerate.

This phenomenon, on the one hand, is due to the loss of the integrated gene, but more importantly, it is undoubtedly caused by the pollution of wild algae and the low purity of transformed algae.

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