Isolation And Functional Analysis Of Genes Related To Carotenoid Metabolism In Pummmelo

As one of natural pigments, carotenoid pigments have a rich nutritional value as well as providing fruits and flowers with distinctive colors. As one of important consistants of carotenoids, lycopene could has important roles, such as anticancer activity. Because of containing two non-hydroxylated β-ionone rings,β-carotene could convert into vitamin A efficiently and supply vitamin Ato mammals. So itwas important to improve carotenoid contents in plants by understanding the molecular metabolism of carotenoid biosynthesis and metabolisms.Major enzymes involved in carotenoid metabolism include PSY, PDS, ZDS, ZISO, CrtISO, LYCB, LYCE and BCH. Except for CrtISO and BCH, all genes had been isolated and analyzed the relative gene expression modelsduringthe fruit development of red-fleshed pummelo and guanxi pummelo. The result showed that few amino acid differences between the two kinds of pummeloes. So, we decided to analyze the expression of genes related to carotenoid metabolism in them. Beside CgZISO, the expression level of upstream genes (CgPSY, CgPDSand CgZDS) which catalyze lycopene synthesistranscriptsincreased during the whole fruit development stage. On the contrary, the downstream genes (CgLYCBand CgLYCE) were decreased.As an upstream gene, CgZISO expression model was similar with downstream genes.Protein-protein interaction of six enzymes in the pathway of carotenoid metabolism was studied by yeast two-hybrid system. The result was similar with BiFCfluorescence microscopy. CgPSY can interact with CgPDS, CgZDS and CgLYCB (CgPSY-CgLYCE failed to turn into yeast), while CgPDS could interact with CgPSY, CgZISO, CgZDS, CgLYCB and CgLYCE. CgZDS showed a similar interaction activity with CgPDS. Compared with other enzymes, CgZISO could interact only with CgPDS and CgZDS, and could not interact with CgPSY, CgLYCB and CgLYCE. The interaction result of CgLYCB and CgLYCE was similar. They could interact with CgPSY, CgPDS and CgZDS. CgLYCB andCgLYCE also had an interaction.A RING-H2 finger gene (CgRHF) was isolated from red-fleshed pummelo and guanxi pummelo. The gene belonged to C3H2C3 type andno difference between two cultivars. Using BiFC, we analyzed the interaction betweenCgRHF and enzymes involved in carotenoid biosynthesis and metabolism. The result showed that CgRHF 1 had an interaction with CgPSY, CgZDS, CgLYCB and CgLYCE isolated from red-fleshed pummelo and guanxi pummelo. CgRHFl only interacted with CgPDS isolated from red-fleshed pummelo. Moreover, we silenced the CgRHF gene in potato using VIGS. The result showed that after CgRHF silenced, the color of potato became pink, and HPLC analysis showed that the carotenoid contents, especially lycopene, β-carotene and lutein, were lower in silenced potato than in negative control. So we deduced that the CgRHF gene might involved in regulating carotenoid biosynthsis and metabolism.A CYP450 gene was isolated from pummelo and had a qPCR analysis. The result showed that CYP450 isolated from pummelo was belonged to CYP86A8 subfamily. qPCR analysis showed that with pummelo became ripe and lycopene accumulation, the gene expression level dramatically decreased following fruit development. CgCYP450 had no interaction with enzymes involving in carotenoid metabolism enzymes. Furthermore, silencedCgCYP450 gene in Nicotiana benthamiana showed a difference in stem compared to negative control.