Zhaodi Li

De novo characterization of the Lycium chinense Mill. leaf transcriptome and analysis of candidate genes involved in carotenoid biosynthesis

Lycium chinense Mill. (Chinese wolfberry), enriching in carotenoids, is an important Chinese herbal medicine. However, studies on the functional genomics research, especially the carotenoid biosynthesis and accumulation, are limited because of insufficiently available datasets. RNA-Seq was performed by the Illumina sequencing platform. Approximately 26 million clean reads were generated after filtering. Clean reads were assembled by SOAPdenovo and subsequently annotated. Among all 61,595 unigenes, 37,816 (61.39%), 25,266 (41.02%), and 17,598 (28.57%) unigenes were annotated in NCBI non-redundant protein, Swiss-Prot, and Kyoto Encyclopedia of Genes and Genomes (KEGG) database, respectively. A total of 16,073 and 11,394 unigenes were assigned to Gene Ontology and Cluster of Orthologous Group, respectively. Furthermore, the majority of genes encoding the enzymes in the carotenoid biosynthesis pathway were identified in the unigene datasets. We first found several genes related to L. chinense carotenoid biosynthesis. The expression levels and the biological functions of these genes involved in carotenoid biosynthesis in the leaf and the green ripening fruit were further confirmed by qPCR and high performance liquid chromatography (HPLC). In the present study, we first characterized the transcriptome of L. chinense leaf, which may provide useful data for functional genomics investigations in L. chinense in the future. And essential genes involved in the carotenoid biosynthesis pathway may contribute to elucidate the expression patterns in different stages of development and fruit ripening and the specific mechanisms of carotenoid biosynthesis/accumulation in L. chinense.

Ectopic expression of the Lycium barbarum β-carotene hydroxylase gene (chyb) enhances drought and salt stress resistance by increasing xanthophyll cycle pool in tobacco

AbstractAbiotic 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 Lycium barbarum β-carotene hydroxylase gene (chyb) under drought and salt stress, we checked the physiological and biochemical properties of chyb-overexpressing transgenic tobacco. 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 suggest that L. barbarum chyb gene dramatically enlarge the xanthophyll cycle pool size, which plays an important role in resistance to drought and salt stress in tobacco.

Overexpression of lycopene ε-cyclase gene from lycium chinense confers tolerance to chilling stress in Arabidopsis thaliana.

Lutein plays an important role in protecting the photosynthetic apparatus from photodamage and eliminating ROS to render normal physiological function of cells. As a rate-limiting step for lutein synthesis in plants, lycopene ε-cyclase catalyzes lycopene to δ-carotene. We cloned a lycopene ε-cyclase gene (Lcε-LYC) from Lycium chinense (L. chinense), a deciduous woody perennial halophyte growing in various environmental conditions. The Lcε-LYC gene has an ORF of 1569bp encoding a protein of 522 aa. The deduced amino acid sequence of Lcε-LYC gene has higher homology with LycEs in other plants, such as Nicotiana tabacum and Solanum tuberosum. When L. chinense was exposed to chilling stress, relative expression of Lcε-LYC increased. To study the protective role of Lcε-LYC against chilling stress, we overexpressed the Lcε-LYC gene in Arabidopsis thaliana. Lcε-LYC overexpression led to an increase of lutein accumulation in transgenic A. thaliana, and the content of lutein decreased when transgenics were under cold conditions. In addition, the transgenic plants under chilling stress displayed higher activities of superoxide dismutase (SOD) and peroxidase (POD) and less H2O2 and malondialdehyde (MDA) than the control. Moreover, the photosynthesis rate, photosystem II activity (Fv/fm), and Non-photochemical quenching (NPQ) also increased in the transgenetic plants. On the whole, overexpression of Lcε-LYC ameliorates photoinhibition and photooxidation, and decreases the sensitivity of photosynthesis to chilling stress in transgenic plants.

Cloning and expression of a ζ-carotene desaturase gene from Lycium chinense

ζ -Carotene desaturase (ZDS) is a key enzyme in the carotenoid biosynthetic pathway which catalyses the conversion of ζ -carotene into lycopene. In this study, a cDNA encoding ZDS was isolated from Lycium chinense by rapid amplification of cDNA ends approach (RACE) and designated as LcZDS. The full-length cDNA sequence was 2042 bp containing a 1767 bp putative open reading frame (ORF) which encoded a 588 amino acid whose molecular weight was 64.69 kDa computationally. The putative protein sequences showed a high degree identity compared with known ZDS from other plants. Phylogenetic analysis depicted that LcZDS has a closer relationship with ZDS of higher plants and chlorophyta than with those of other species. Quantitative real-time PCR assay revealed that the expression of LcZDS was the highest in maturing fruits and was upregulated by different abiotic stresses in L. chinenese. In addition, expression of this gene in Escherichia coli produced a single polypeptide which could successfully catalyze ζ -carotene into lycopene via neurosporene. Carotenoids are structurally diverse group of natural pigments synthesized by all higher plants, algae, some bacteria and fungi (Sandmann 2001). In higher plants, carotenoids represent a class of yellow, orange and red lipid-soluble pigments, providing distinct colours and nutritional quality to flowers, fruits and vegetables. Carotenoids participate in light-harvesting processes and protect photosynthetic apparatuses from photooxidation (Tracewell et al. 2001; Szabó et al. 2005; Dong et al. 2007). Carotenoids can also confer resistance to abiotic stress in plants by scavenging reactive oxygen species (Kim et al. 2012). In animals, carotenoids are indispensable components of antioxidant systems (Palozza

Cloning and characterization of a novel β-carotene hydroxylase gene from Lycium barbarum and its expression in Escherichia coli.

Lycium barbarum contains high levels of zeaxanthin, which is produced by the conversion of β ‐carotene into zeaxanthin. β ‐Carotene hydroxylase catalyzes this reaction. We cloned a cDNA (chyb) encoding β ‐carotene hydroxylase (Chyb) 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. A 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. Thus, this study suggests that L. barbarum β ‐carotene hydroxylase could be a means of zeaxanthin production by genetic manipulation in E. coli.