Cloning And Functional Expression Of The Crucial Genes In Carotenoid Biosynthetic Pathway From Lycium Chinense

Carotenoids are a structurally diverse group of natural pigments. In higher plants, carotenoids participate in light-harvesting processes, protect photosynthetic apparatuses from photooxidation, or act as precursors to plant hormone abscisic acid(ABA). In human and animals, carotenoids serving as important antioxidents and dietary complements of provitamin A, are essential nutrition for human and animals health. Lycium chinense is rich in carotenoids and has great capability of environmental adaption. But until now, studies on the carotenoid biosynthesis genes from L. chinense have not been reported.Six key genes, isopentenyl pyrophosphate isomerase gene(LcIPI), phytoene synthase gene(LcPSY), phytoene desaturase gene(LcPDS), δ-carotene desaturase gene(LcZDS), carotenoid isomerase gene(LcCRTISO) and lycopene ε-cyclase gene(LcLYCE) were firstly cloned from L. chinense by 3’RACE PCR. Bioinformatics analyses discovered that these enzymes were highly conserved in plants and shared similar evolutionary relationship.Color complementation assay and high-performance liquid chromatography(HPLC) analysis were conducted to investigate the catalytic activity of the enzymes. The results strongly suggested that Lc IPI could catalyze isomerization between isopentenyl diphosphate and dimethylallyl diphosphate; LcPSY could catalyze the condensation of two molecules of geranylgeranyl diphosphate to phytoene; LcPDS could catalyze two desaturation steps from phytoene to δ-carotene; LcZDS could catalyze δ-carotene into lycopene and Lc LYCE could catalyze cyclization of lycopene to lutein. The enzymes obtained here all have catalytic activity.Transgenic tobaccos including LcPSY, LcPDS, LcZDS, LcLYCE and LcCRTISO respectively, were successfully obtained by Agrobacterium-mediated genetic transformation system. Transgenic tobaccos showed enhanced content of carotenoids in leaves to different extents compared with control as the following order: LcPSY>LcLYCE>LcCRTISO>LcPDS>LcZDS.Salt stress is one of the major environmental factors restricting the productivity and distribution of crops worldwide. Carotenoids play a positive role in plant stress resistance. Due to the massive carotenoids accumulation of transgenic tobacco overexpressing LcPSY, we investigated the stress resistance of the LcPSY. Compared with the control plant, the transgenic lines exhibited larger values of photosynthesis rate(Pn) and photosystem II activity(Fv/Fm), higher activities of superoxide dismutase(SOD) and peroxidase(POD), and lower accumulation of H2O2 and O2-under salt stress. These results indicated that overexpressing LcPSY in tobacco successfully improved its salt tolerance by increasing the carotenoid biosynthetic capacity, which impaired ROS production, protected plants from oxidative stress.