Cloning And Research Of β-carotenoid Hydroxylase Gene And Its Promoter Of Lycium Barbarum

Carotenoids are naturally occurring fat-soluble and pigmented compounds because of their structure and desaturation level, synthesized by fungi, bacteria, and plants. Owing to their role in photosynthesis, carotenoids are ubiquitous in photosynthetic organisms. Carotenoids also perform essential functions within non-photochemical quenching of reactive oxygen species and preventing DNA damage.Beta-carotene hydroxylases catalyze the hydroxylation of β-carotene to form zeaxanthin, which is a kind of xanthophyll. Lycium barbarum is a kind of traditional Chinese medicine with many important biological activities, such as anticancer, antiaging, and antioxidation. However, no studies have been conducted to clone the β-carotene hydroxylase gene(chyb) from L. barbarum. This dissertation is aimed to clone and characterize the L. barbarum chyb gene by molecular, bioinformatics, and physiological measurements of transgenic tobacco plants in resistance to abiotic stress.Firstly, a cDNA(chyb) encoding β-carotene hydroxylase(CHYB) was cloned from the L. barbarum leaf. A 939-bp full-length cDNA sequence was determined with 3’-rapid amplification of cDNA end assay encoding a deduced CHYB protein(34.8 kDa) with a theoretical isoelectric point of 8.36. The results of bioinformatics analysis showed that the L. barbarum CHYB was located in the chloroplast. Further to investigate the catalytic activity of the L. barbarum CHYB, a complementation analysis was conducted in Escherichia coli. The results strongly demonstrated that CHYB can catalyze β-carotene to produce zeaxanthin. So this study provides a new way for the biosynthesis of zeaxanthin.Then, abiotic stresses, such as drought and salinity, are major limiting factors for plant growth and reproduction. Carotenoids play key roles in response to abiotic stresses. To identify the role of L. barbarum β-carotene hydroxylase gene under drought and salt stress, the physiological and biochemical properties of chyb-overexpressing transgenic tobacco were checked in this study. Ectopic expression of chyb in tobacco increased the drought and salt tolerance in the transgenic lines. The amounts of xanthophylls cycle pigments were significantly increased in the transgenic lines compared with the controls. Under osmotic stress conditions, the root length and shoot biomass of the transgenic lines were significantly increased. Under drought stress conditions, the transgenic lines leaves lost water slower than the WT plants, and the photosynthesis rate and SOD activity were significantly increased, whereas stomatal conductance and malondialdehyde content were decreased in the transgenic lines. Under salt stress conditions, proline and chlorophyll levels of the transgenic lines were significantly increased, whereas malondialdehyde and Na+/K+ ratios were significantly decreased. Thus, these findings suggested that L. barbarum chyb gene dramatically enlarged the xanthophyll cycle pool size, which plays an important role in resistance to drought and salt stress in tobacco. So this study provides important candidate genes for plant drought and salt tolerant molecular breeding.Furthermore, in order to research the function of L. barbarum chyb gene promoter(chybpro) in the gene expression regulation, the promoter of chyb gene were cloned and studied. Bioinformatics analysis results indicated that there were many light responsive and endosperm expression elements in chybpro sequence. It was implied that L. barbarum chybpro may be used for light response and endosperm specificity expression in plant.