Ectopic overexpression of a type-II DGAT (CeDGAT2-2) derived from oil-rich tuber of Cyperus esculentus enhances accumulation of oil and oleic acid in tobacco leaves.

BACKGROUND: Engineering triacylglycerol (TAG) accumulation in vegetative tissues of non-food crops has become a promising way to meet our increasing demand for plant oils, especially the renewable production of biofuels. The most important target modified in this regard is diacylglycerol acyltransferase (DGAT) enzyme responsible for the final rate-limiting step in TAG biosynthesis. Cyperus esculentus is a unique plant largely accumulating oleic acid-enriched oil in its underground tubers. We speculated that DGAT derived from such oil-rich tubers could function more efficiently than that from oleaginous seeds in enhancing oil storage in vegetative tissues of tobacco, a high-yielding biomass crops. RESULTS: Three CeDGAT genes namely CeDGAT1, CeDGAT2-1 and CeDGAT2-2 were identified in C. esculentus by mining transcriptome of developing tubers. These CeDGATs were expressed in tissues tested, with CeDGAT1 highly in roots, CeDGAT2-1 abundantly in leaves, and CeDGAT2-2 predominantly in tubers. Notably, CeDGAT2-2 expression pattern was in accordance with oil dynamic accumulation during tuber development. Overexpression of CeDGAT2-2 functionally restored TAG biosynthesis in TAG-deficient yeast mutant H1246. Oleic acid level was significantly increased in CeDGAT2-2 transgenic yeast compared to the wild-type yeast and ScDGA1-expressed control under culture with and without feeding of exogenous fatty acids. Overexpressing CeDGAT2-2 in tobacco led to dramatic enhancements of leafy oil by 7.15- and 1.7-fold more compared to the wild-type control and plants expressing Arabidopsis seed-derived AtDGAT1. A substantial change in fatty acid composition was detected in leaves, with increase of oleic acid from 5.1% in the wild type to 31.33% in CeDGAT2-2-expressed tobacco and accompanied reduction of saturated fatty acids. Moreover, the elevated accumulation of oleic acid-enriched TAG in transgenic tobacco exhibited no significantly negative impact on other agronomic traits such as photosynthesis, growth rates and seed germination except for small decline of starch content. CONCLUSIONS: The present data indicate that CeDGAT2-2 has a high enzyme activity to catalyze formation of TAG and a strong specificity for oleic acid-containing substrates, providing new insights into understanding oil biosynthesis mechanism in plant vegetative tissues. Overexpression of CeDGAT2-2 alone can significantly increase oleic acid-enriched oil accumulation in tobacco leaves without negative impact on other agronomy traits, showing CeDGAT2-2 as the desirable target gene in metabolic engineering to enrich oil and value-added lipids in high-biomass plants for commercial production of biofuel oils.

Screening a specific Zn(ii)-binding peptide for improving the cognitive decline of Alzheimer's disease in APP/PS1 transgenic mice by inhibiting Zn2+-mediated amyloid protein aggregation and neurotoxicity.

Zn2+ has been implicated in the progression of Alzheimer's disease (AD), as amyloid-ß protein (Aß) aggregation and neurotoxicity are mediated by zinc ions. Therefore, development of metal chelators for inhibiting and regulating metal-triggered Aß aggregation has received attention as a strategy for treating AD. Here, we used an approach based on phage display to screen for a Zn(ii)-binding peptide that specifically blocks Zn-triggered Aß aggregation. A fixed Zn(ii) resin was prepared using Ni-IDA affinity resin, and the target Zn(ii) was screened by interaction with a heptapeptide phage library. After negative biopanning against IDA and four rounds of positive biopanning against Zn(ii), high specificity Zn(ii)-binding phages were obtained. Through DNA sequencing and ELISA, 15 sets of Zn(ii)-binding peptides with high histidine contents were identified. We chose a highly specific peptide against Zn(ii) with the sequence of H-M-Q-T-N-H-H, and its abilities to chelate Zn2+ and inhibit Zn2+-mediated Aß aggregation were assessed in vitro. We loaded the Zn(ii)-binding peptide onto PEG-modified chitosan nanoparticles (NPs) to improve the stability and the bioavailability of the Zn(ii) binding peptide. PEG-modified chitosan NPs loaded with Zn(ii)-binding peptide (PEG/PZn-CS NPs) reduced Zn2+ concentrations and Aß secretion in mouse neuroblastoma (N)2a cells stably over-expressing the APP Swedish mutation (N2aswe). Zn2+-Induced neurotoxicity, oxidative stress, and apoptosis were attenuated by PEG/PZn-CS NPs. Intranasal administration of PEG/PZn-CS NPs improved the cognitive ability of APPswe/PS1d9 (APP/PS1) double-transgenic mice and reduced Aß plaques in the mouse brain. This study indicated that a Zn(ii)-binding peptide and its NPs have promise as a potential anti-AD agent.

Localization of ZnT7 and zinc ions in mouse retina--immunohistochemistry and selenium autometallography.

Zinc transporter 7 (ZnT7, Slc30a7), a member of the Slc30 family, is involved in mobilizing zinc ions from the cytoplasm into the Golgi apparatus. In the present study, we examined the distribution and localization of ZnT7 and the labile zinc ions in the mouse retina using immunohistochemistry and in vivo zinc-selenium autometallography (ZnSe(AMG)). Our results showed that ZnT7 is abundantly expressed in the ganglion cells and pigment epithelial cells of the mouse retina. ZnT7 is also expressed in the amacrine cells and the layer of optic fibers of the mouse retina, but to a lesser extent. Weak staining of ZnT7 was detected in the inner plexiform layer, outer plexiform layer, and outer segment of the photoreceptors. However, ZnT7 was not detected in the outer nuclear layer and inner segment of the photoreceptors. A high level of labile zinc pool was detected in the pigment epithelial cells, the inner segment of the photoreceptors, and the marginal region of the inner nuclear layer. Less amount of labile zinc ions were detected in the ganglion cells of the retina. These observations strongly suggest that ZnT7 may play critical roles in retinal zinc homeostasis and that chelatable zinc pools may have multiple functions in the retina.