Breeding Of High Lipid Algae And DGAT2 Gene Function

Microalgae can live widely on land and in sea. They are lower autotrophic plants, and their greatest feature are the wide variety of widely distributed and breeding fast. Microalgae is very rich in glycerin and esters, so microalgae can be used as good raw material for biodiesel. The prerequisites have the good algae species and optimal culture conditions for the development and use of oil-producing microalgae. They have become research topics of common concern at home and abroad.There is a rich algae species in the natural environment resources. Therefore the most direct way is to select the ideal algae strains from the environment. We selected the high-oil algae strains Micr-actinium pusillum Y-002 as the experimental material. It is classified by morphological and molecular biological method of combining on the species.Medium composition has great influence on the algae oil production. We study four different medias (TAP, HSM, SE, and BG11) and limited nutrient as culture conditions to cultivate Micractinium pusillum Y-002. We observed the growth and neutral lipid accumulation of microalgaes. The results showed that:when the nitrogen-free culture, the neutral lipid content of microalgae have raised(2-8 times) in different media. The content of neutral lipid in HSM-N medium is more than ten times higher than in the general HSM. The iron-free culture only little effect on the neutral lipid. And when it is nutrient-free, it will significantly reduce the growth of biomass.It is another way to find the ideal microalgae algae strains through physical, chemical and biological methods such as artificial mutation. In this study, Chlamydomonas reinhardtii CC425 is insertional mutagenesis to search for the ideal algae strains. We selected a mutant CC425-M23. The neutral lipid content increases in HSM and HSM-N media. In HSM, CC425-M23 mutant is compared with the wild-type CC425 the neutral lipid content increased by 170%. It is 17% of dry cell weight. The neutral lipid content of mutant were increased by 14% in HSM-N.The third screening method is the use bio-genetic engineering and metabolic engineering of biological to " artificially create "project microalgae. We clone the key gene fragments of CrDGAT genes in TAG biosynthesis, and constructed CrDGAT gene RNAi expression vector. Then we put it transformed into Chlamydomonas reinhardtii CC425. The results showed that the neutral lipid is significantly reduced while only silence CrDGAT-1 and CrDGAT-5 gene in the predicted 5 CrDGAT genes. The neutral lipid content decreased by 15% and 27% in HSM, and the transformants death in HSM-N. This shows CrDGAT-1 and CrDGAT-5 gene is the real synthesis and expression rate-limiting enzyme genes of TAG.We build CrDGAT gene overexpression vector, overexpression CrDGAT-1 and CrDGAT-5 gene, and the subcellular localization of CrDGAT gene. As a result, the neutral lipid content of the transformants compared with wild-type CC425 were increased by 20%-40%, the largest neutral lipid content was 59% and 50% in the HSM-N. CrDGAT Subtype cellular localization showed that:CrDGAT-1 and CrDGAT-5 gene is expressed in the cytoplasm. At present, breeding of microalgae are mainly three ways above. This study, we use three methods to find the ideal types of oil-producing algae. And explore the possible mechanism and location of the CrDGAT genes. This can lay the foundation for further study the mechanism of oil-producing microalgae.