Screening Of Tropical Microalgae With High Neutral Lipids Content And The Effect Of Pepc2 Gene Silencing On Microalgal Lipid Accumulation

Microalgae usually refer to the photosynthetic microbes containing chlorophyll a (including Cyanobacteria). As a new biodiesel raw material with considerable prospect, microalgae has a lot of advantages, such as wide variety of sources, low cost, clean, renewable, and so on. There are some common concern of the international about microalgae biodiesel, such as screening of lipid-rich algae species, exploring the best culture conditions and construction of genetic engineering algae containing numerous lipid by use of biotechnology.In this experiment, a large number of algal species in the tropical waters have been isolation. Interesting algae strains have been classified through the combination of morphological and molecular biology methods. Isolation and identification of algal species prepare well for the further physiological and biochemical analysis, molecular biology and genetics study of microalgae; and abundant algae species resource can lay a good foundation for high-throughput screening of lipid-rich microalgae.By Nile Red fluorescence staining method, microalgae lipid can be qualitatively and quantitatively analyzed. We optimized the detection conditions:Nile red dye concentration 0.5μg/ml, tested algae concentration OD490=0.1-1.0, DMSO concentration 20% and the dyeing time 10min. This provides a fast, sensitive and convenient experimental method for high-throughput screening of lipid-rich microalgae.The growth and lipid accumulation of three kinds of Chlorella vulgaris were tested in four different media (SE, BG11, TAP, DS-1) culture conditions and a tubular photobioreactor for microalgae was produced. They laid the foundation for large-scale and high-density training of microalgae. The results showed that, TAP medium was more suitable for high-density culture of Chlorella, while the DS was more suitable for lipid accumulation. And we screened algae species Y019 with the ideal lipid accumulation. According to the relationship between medium composition and lipid content, we speculate that the excessive lipid accumulation of microalgae is one kind of stress physiological response to resist adverse environment through the accumulation of storage material.A Pepc2 gene fragment was cloned, which is a key gene in lipid synthesis and metabolism. Pepc2 gene RNAi expression vector was constructed, and transformed into Chlamvdomonas reinhardtii CC-124 wild type mt-by glass bead method. The transcription of Pepc2 gene was analyzed by Fluorescent Quantitative PCR. The results showed that RNAi led to high levels of Pepc2 gene silencing. Then, we analyzed the lipid content of transgenic algae using Nile Red fluorescence staining method. Compared with the control, the lipid content of transgenic algae was up to 23.4%.