Optical Technique For Genome Shuffling

Genome shuffling is a new technology in breeding, which is widely used to obtain high-yield strains of microorganisms. Genome shuffling is a process that combines the advantage of multi-parental crossing with the recombination of entire genomes by cell fusion. The improved phenotype is selected by multi-round recursive cell fusion in hybrid cells with positive character.The effective fusion is the key in genome shuffling technology. At the present, genome shuffling technology is based on chemical agent induced cell fusion at population level, which is an uncontrolled and non-selective operation. If the technology can be operated at single cell level, it will be a great improvement. Cell fusion between selected cells and screening of hybrid cells with positive character is required for genome shuffling technology at single cell level. Precise operation, sensitive detection and accurate analysis will be needed in this process.Astaxanthin is a kind of carotenoids with strong anti-oxidation function. Because of limited sources, the production of natural astaxanthin is far from meeting the current need. Thus, the selection and breeding of strains with high-yield become an extremely important issue.In this paper femtosecond laser-induce protoplast fusion was studied to establish a new method of single cell level genome shuffling and to obtain astaxanthin high- product strain. The femtosecond laser microscopy manipulation system was utilized as the experimental platform. The Haematococcus pluvialis and Phaffia rhodozyma cells, the main sources of natural astaxanthin, were the objective of the research. And several kinds of spectrometry were used to select the high-yielding strains.The main contents in this paper are as follows:(1) The effect of femtosecond laser on Phaffia yeast cells and protoplasts were studied. Femtosecond laser-induced cell fusion with Phaffia rhodozyma protoplast was achieved and a hypothesis model of femtosecond laser-induce cell fusion was presented.(2) Preliminary results were got from the optical trapping using the photonic crystal fiber. And the hollow mode was successfully obtained though filling the air hole of the photonic crystal fiber with the material whose refractive index higher than the quartz. The hollow beam will cause less damage to cells and has important meaning to the research of optical trapping. And the method to obtain hollow beam is a very simple way to transform optical mode.(3) The characteristics Raman spectrum and Terahertz "fingerprint" spectrum of Phaffia rhodozyma and Haematococcus pluvialis were got. And a set of two-photon fluorescence detecting system using femtosecond laser was established. The fluorescence of chlorophyll was detected using this system.