Preliminary Study On Pseudochlorococcum Expressing Vitreoscilla Hemoglobin And Overexpression Of C4-like Pathway CyPPDK Gene

Nannochloropsis is a kind of marine unicellular microalgae.As a highly efficient photosynthetic cell factory,it has great commercial value in the biotechnology field of biofuels and high value-added products.During the growth process,excessive reactive oxygen species(ROS)cause by intense light or high temperature in microalgal cells resulted in a decrease of cell growth rate and unsaturated fatty acid content.In recent years,genetic engineering has been carried out to improve the robustness of microalgae for yields of biomass and value-added products.The object of this study is to screen the strong endogenous promoters from N.oceanica.The exogenous VHb gene from Vitreoscilla is expressed in N.oceanica to relieve the hurt from ROS,and the endogenous cyPPDK gene is overexpressed.(1)Based on comparative transcriptome analysis,the 1000 bp upstream of three genes(LHC,HSP20 and RPL32)with higher FPKM value were selected as promoter candidates of Nannochloropsis.An expression vector including one of the above three promoters,GFP and zeocin gene was used to test the test the efficiency of these promoters.The expression levels of the GFP gene promoted by RPL32,LHC and HSP20 showed that they were 1.15,5.46 and 6.58 times higher than reference Actin gene,respectively,indicating that they are the promising promoter candidates for microalgae genetic engineering.(2)The Vitreoscilla hemoglobin gene(VHb)is transcribed and directed into the chloroplasts of N.oceanica IMET1 using the LHC promoter and signal peptide sequence of the LHC gene.The activity of hemoglobin was verified by CO-differential spectroscopy.Under the condition of high light stress,the growth rate of the optimal transformant was increased by 24%,the EPA content was increased by 34% compared with the wide type,and the Fv/Fm was significantly higher than that of the wild type.This indicates that VHb can reduce oxidative damage and improve the growth of microalgae under stress conditions.(3)The endogenous gene cyPPDK of Nannochloropsis was overexpressed with HSP20 promoter.PCR results showed that expression vector was successfully electroprated into the cells,and the resistance was not lost after 12 passages of subculture.RT-PCR analysis demonstrated that the expression level of the gene related to the C4 pathway in the transformants was up-regulated compared with the wild type.In addition,the optimal transformant had a 32% higher lipid content than the wild type strain.The cyPPDK gene plays an important role in promoting C4-like cycle and enhance the lipidaccumulation by distributing substances and balancing energy metabolism.