The lineage-specific evolution of the oleosin family in Theaceae.

Oleosins play essential roles in stabilization of lipid droplets (LDs) and seed oil production. However, evolution of this gene family has not been reported in Theaceae, a large plant family that contains many important tea and oil tea species. In this study, a total of 65 oleosin genes were identified in nine genome-sequenced Theaceae species. Among these genomes, the gene number of oleosin showed significant difference, with Camellia sinensis var. sinensis cv. Shuchazao and Camellia lanceoleosa displayed more oleosin numbers than other species. Phylogenetic analyses revealed that Theaceae oleosin genes were classified into three clades (U, SL, SH) respectively. Proteins within the same clade had similar gene structure and motif composition. Segmental duplication was the primary driving force for the evolution of oleosin genes in Shuchazao (SCZ), Huangdan (HD), C.lanceoleosa (Cla), and wild tea (DASZ). Synteny analysis showed that most oleosin genes displayed inter-species synteny among tea and oil tea species. Expression analysis demonstrated that oleosin genes were specifically expressed in seed and kernel of Huangdan (HD) and C.lanceoleosa. Moreover, expression divergence was observed in paralogous pairs and ∼1-2 oleosin genes in each clade have become activate. This study leads to a comprehensive understanding of evolution of oleosin family in Theaceae, and provides a rich resource to further address the functions of oleosin in tea and oil tea species.

Expansion and Diversification of the 14-3-3 Gene Family in Camellia sinensis.

14-3-3 proteins are signal moderators in sensing various stresses and play essential functions in plant growth and development. Although, 14-3-3 gene families have been identified and characterized in many plant species, its evolution has not been studied systematically. In this study, the plant 14-3-3 family was comprehensively analyzed from green algae to angiosperm. Our result indicated that plant 14-3-3 originated during the early evolutionary history of green algae and expanded in terricolous plants. Twenty-six 14-3-3 genes were identified in the tea genome. RNA-seq analysis showed that tea 14-3-3 genes display different expression patterns in different organs. Moreover, the expression of most tea 14-3-3 genes displayed variable expression patterns under different abiotic and biotic stresses. In conclusion, our results elucidate the evolutionary origin of plant 14-3-3 genes, and beneficial for understanding their biological functions and improving tea agricultural traits in the future.

Genome-wide identification and expression analysis of the MADS-box transcription factor family in Camellia sinensis.

The MADS-box genes are an important class of transcription factors and play critical roles in flower development. However, the functions of these genes in the economically important drinking plant, Camellia sinensis, are still not reported. Here, an evolutionary analysis of tea MADS-box genes was performed at whole genome level. A total of 83 MADS-box genes were identified in tea, and their gene structures and expression patterns were further analyzed. The tea MADS-box genes were classified into Mα (26), Mß (12), Mγ (9), MIKC* (7), and MIKCC (29) clade according to their phylogenetic relationship with Arabidopsis thaliana. Several cis-elements were identified in the promoter regions of the CsMADS genes that are important in regulating growth, development, light responses, and the response to several stresses. Most CsMADS genes display clear different expression patterns in different organs and different species of tea plant. The expression of CsMADS genes can be regulated by abiotic stresses and phytohormone treatment. Our results lay the foundation for future research on the function of CsMADS genes and beneficial for improving tea agricultural traits in the future.

Daily rhythmicity of clock gene transcript levels in fast and slow muscle fibers from Chinese perch (Siniperca chuatsi).

BACKGROUND: Clock genes are considered to be the molecular core of biological clock in vertebrates and they are directly involved in the regulation of daily rhythms in vertebrate tissues such as skeletal muscles. Fish myotomes are composed of anatomically segregated fast and slow muscle fibers that possess different metabolic and contractile properties. To date, there is no report on the characterization of the circadian clock system components of slow muscles in fish. RESULTS: In the present study, the molecular clock components (clock, arntl1/2, cry1/2/3, cry-dash, npas2, nr1d1/2, per1/2/3, rorα and tim genes) and their daily transcription levels were characterized in slow and fast muscles of Chinese perch (Siniperca chuatsi). Among the 15 clock genes, nrld2 and per3 had no daily rhythmicity in slow muscles, and cry2/3 and tim displayed no daily rhythmicity in fast muscles of the adult fish. In the slow muscles, the highest expression of the most clock paralogs occurred at the dark period except arntl1, nr1d1, nr1d2 and tim. With the exception of nr1d2 and tim, the other clock genes had an acrophase at the light period in fast muscles. The circadian expression of the myogenic regulatory factors (mrf4 and myf5), mstn and pnca showed either a positive or a negative correlation with the transcription pattern of the clock genes in both types of muscles. CONCLUSIONS: It was the first report to unravel the molecular clock components of the slow and fast muscles in vertebrates. The expressional pattern differences of the clock genes between the two types of muscle fibers suggest that the clock system may play key roles on muscle type-specific tissue maintenance and function.

Recombinant keratinocyte growth factor 1 in tobacco potentially promotes wound healing in diabetic rats.

Keratinocyte growth factor 1 (KGF1) is a growth factor that promotes epidermal cell proliferation, migration, differentiation, and wound repair. It is expressed at low levels in a form of inclusion body in E. coli. In order to increase its expression and activity, we produced tobacco plants expressing KGF1 via Agrobacterium-mediated transformation using a potato virus X (PVX)-based vector (pgR107). The vector contained the sequence encoding the KGF1 gene fused with a green florescence protein. The recombinant plasmid was introduced into leaf cells of Nicotiana benthamiana (a wild Australian tobacco) via Agrobacterium-mediated agroinfiltration. As determined by fluorescence and Western blot of leaf extracts, the KGF1 gene was correctly translated into the tobacco plants. The recombinant KGF1 was purified from plant tissues by heparin affinity chromatography, and cell proliferation in NIH/3T3 cells was stimulated by the purified KGF1. The purified KGF1 was also applied to the wounds of type-II diabetic rats. KGF1 had accumulated to levels as high as 530 µ g/g fresh weight in the leaves of agroinfected plants. We show that plant-derived KGF1 can promote the proliferation of NIH/3T3 cells and have significant effects on the type-II diabetic rat. The present findings indicated that KGF1 from tobacco maintains its biological activity, implying prospective industrial production in a plant bioreactor.

Selection of high efficient transdermal lipid vesicle for curcumin skin delivery.

Curcumin shows effective anti-inflammatory activities but is seldom used in clinic because of its poor solubility in water and vulnerablity to sunshine ultraviolet effect. Novel lipid vesicles have been developed as carriers for skin delivery. In this paper, lipid vesicles-propylene glycol liposomes (PGL), Ethosomes and traditional liposomes, were prepared as curcumin carriers respectively. Their morphology, particle size and encapsulation efficiency and drug release behavior in vitro were evaluated. Transdermal efficiency and deposition quantity in abdominal skin were also measured with Franz diffusion device. Carrageenan-induced paw edema was established to evaluate the anti-inflammatory effect. From the result, the particle size order of lipid vesicles was: PGL (182.4 ± 89.2 nm)Ethosomes>traditional liposomes. PGL had the best encapsulation efficiency of 92.74 ± 3.44%. From anti-inflammatory experiment, PGL showed the highest and longest inhibition on the development of paw edema, followed by Ethosomes and Traditional liposomes. With the elevated entrapment efficiency, good transdermic ability and sustained-release behavior, PGL may represent an efficient transdermal lipid vesicle for skin delivery.

A novel cold-regulated gene from Camellia sinensis, CsCOR1, enhances salt- and dehydration-tolerance in tobacco.

In present research, the full-length cDNA and the genomic sequence of a novel cold-regulated gene, CsCOR1, were isolated from Camellia sinensis L. The deduced protein CsCOR1 contains a hydrophobic N-terminus as a signal peptide and a hydrophilic C-terminal domain that is rich in glycine, arginine and proline. Two internal repetitive tridecapeptide fragments (HSVTAGRGGYNRG) exist in the middle of the C-terminal domain and the two nucleotide sequences encoding them are identical. CsCOR1 was localized in the cell walls of transgenic-tobaccos via CsCOR1::GFP fusion approach. The expression of CsCOR1 in tea leaves was enhanced dramatically by both cold- and dehydration-stress. And overexpression of CsCOR1 in transgenic-tobaccos improved obviously the tolerance to salinity and dehydration.

Expression, purification, and characterization of functional recombinant human daintain/AIF-1 in Escherichia coli.

Daintain/AIF-1 was identified from injured rat carotid arteries and porcine intestine in the mid 1990s. It is involved in autoimmune disorders, chronic rejection of allografts, gliomas, and breast cancer. Since it is convenient and economical to obtain such a peptide biologically, in this study, we describe the expression, purification, and characterization of recombinant human daintain/AIF-1 (rhdaintain/AIF-1). The backbone of vector pET32a, a high-level expression plasmid, was used to construct the pET32a-daintain/AIF-1 plasmid for daintain/AIF-1 expression in Escherichia coli. The recombinant daintain/AIF-1 protein was solubly expressed in the BL21 (DE3) strain and was purified by Ni2+ affinity chromatography. After purification, the recombinant protein showed the expected size of 18 kDa on Tricine-SDS-PAGE gels which was further confirmed by Western blotting. A total of 34.0 mg of high purity (over 98%) rhdaintain/AIF-1 was obtained from 1 L culture. The recombinant peptide was able to increase blood glucose elimination rates and enhance the proliferation of human MCF-7 cells. These results suggest that biological activity of the recombinant peptide was preserved after purification.