Study On The Synthesis Of Polyunsaturated Fatty Acids By Chromosomal B5 And Its Reductase

Polyunsaturated fatty acids (PUFAs) are essential components for many cellular and physiological processes. Organism required a balance of PUFAs, which affects the normal physiological functions. In mammals, PUFAs are synthetized via an aerobic process from saturated fatty acid precursors by a muti-enzymes system involving cytochrome b5, flavoprotein-NADH-dependent cytochrome b5 reductase (Ncb5or), and fatty acid desaturases. Despite sporadic reports showing the association of certain fatty acid deficiencies with NADH-cytochrome b5 reductases or cytochrome b5 in mouse, leishmania major, and arabidopsis, the knowledge about the relationship between NADH-cytochrome B5 reductase, cytochrome b5, stearoyl-CoA desaturase and some other desaturases in PUFAs synthesis is poor. It’s unclear what NADH-dependent cytochrome b5 reductase and cytochrome b5 existing in C.elegans. It was not clear that do from FAT-1 to FAT-7 all of seven desaturases need the same or distinct flavoprotein-NADH-dependent cytochrome b5 reductase and cytochrome b5 to be activated in synthetizing PUFAs?In this study, C.elegans as a model organism, which use to explore the mechanisms that NADH-cytochrome B5 reductases and cytochrome b5, belongs to the super family of P450 enzymes genes affect the PUFAs synthesis by the methods of molecular biology and genetics. RNAi-mediated suppression was performed on NADH-cytochrome B5 reductases and cytochrome b5 and evaluated the resulting fatty acids composition with GC. Fat content determined by TLC/GC. Quantitative real-time PCR results demonstrated the expression of key fatty acids synthesis and decomposition genes, the fluorescence intensity of over-expression and with GFP showed the expression of its protein, dietary supplementation to suppress the defects phenotypes.In this study, it was been found NADH-dependent cytochrome b5 reductase and cytochrome b5, as well as characterize their role for the activity of different desaturases in C.elegans. And was been found RNAi-mediated suppression of NADH-dependent cytochrome b5 reductase (T05H4.4/hpo-19) and cytochrome b5(cytb-5.1) result in significant changes in fatty acid composition in worms. Inactive of hpo-19 and cytb-5.1 led to a variety of abnormal phenotypes such as small lipid droplet size, reduced brood sides, few PUFAs content and fat content decreased. In addition, hpo-19 null worms show very slow growth rate than control, the lifespan of cytb-5.1 null worms was significantly shorter than control. It was been found that during desaturases gene expression, the fat-2, fat-6 and fat-7 increased significantly in hpo-19 and cytb-5.1. Dietary supplementation can supress these defect phenotypes. When adding C18:3n3, C18:3n6 and C20:4n6 supplementary can rescue their production respectively, but When adding C20:3n6, these fatty acids accumulated in worms and can’t convert into 20:4n6 in hpo-19 worms. On the other hand, all 6 fatty acids that C18:1n9, C18:2n6, C18:3n3, C18:3n6, C20:3n6 and C20:4n6 supplementary were sufficient to rescue their production and the phenotypes of cytb-5.1 deficient worms. Cytochrome b5 (CYTB5.1) mainly acts as a partner of FAT-6 and FAT-7 to govern fatty acids metabolism. Meanwhile, cytochrome b5 reductase (HPO-19) is work together with FAT-1, FAT-2, FAT-3 and FAT-4, mainly FAT-2 and FAT-4 to regulate worms PUFAs metabolism.