Persistent Targeting DNA Nanocarrier Made of 3D Structural Unit Assembled from Only One Basic Multi-Palindromic Oligonucleotide for Precise Gene Cancer Therapy.

Construction of a simple, reconfigurable, and stimuli-responsive DNA nanocarrier remains a technical challenge. In this contribution, by designing three palindromic fragments, a simplest four-sticky end-contained 3D structural unit (PS-unit) made of two same DNA components is proposed. Via regulating the rotation angle of central longitudinal axis of PS-unit, the oriented assembly of one-component spherical architecture is accomplished with high efficiency. Introduction of an aptamer and sticky tail warehouse into one component creates a size-change-reversible targeted siRNA delivery nanovehicle. Volume swelling of 20 nm allows one carrier to load 1987 siPLK1s. Once entering cancer cells and responding to glutathione (GSH) stimuli, siPLK1s are almost 100% released and original size of nanovehicle is restored, inhibiting the expression of PLK1 protein and substantially suppressing tumor growth (superior to commercial transfection agents) in tumor-bearing mice without systemic toxicity.

Intelligent Reconfiguration-Promoted Cellular Internalization of Core-Shell DNA Nanoprobe Equipped with Successive Dual Stimuli-Responsive Protective Satellites for Amplification Fluorescence Imaging of Tumor Cells.

Although DNA probes have attracted increasing interest for precise tumor cell identification by imaging intracellular biomarkers, the requirement of commercial transfection reagents, limited targeting ligands, and/or non-biocompatible inorganic nanostructures has hampered the clinic translation. To circumvent these shortcomings, a reconfigurable ES-NC (Na+ -dependent DNAzyme (E)-based substrate (S) cleavage core/shell DNA nanocluster (NC)) entirely from DNA strands is assembled for precise imaging of cancerous cells in a successive dual-stimuli-responsive manner. This nanoprobe is composed of a strung DNA tetrahedral satellites-based protective (DTP) shell, parallelly aligned target-responsive sensing (PTS) interlayer, and hydrophobic cholesterol-packed innermost layer (HCI core). Tetrahedral axial rotation-activated reconfiguration of DTP shell promotes the exposure of interior hydrophobic moieties, enabling cholesterol-mediated cellular internalization without auxiliary elements. Within cells, over-expressed glutathione triggers the disassembly of the DTP protective shell (first stimulus), facilitating target-stimulated signal transduction/amplification process (second stimuli). Target miRNA-21 is detected down to 10.6 fM without interference from coexisting miRNAs. Compared with transfection reagent-mediated counterpart, ES-NC displays a higher imaging ability, resists nuclease degradation, and has no detectable damage to healthy cells. The blind test demonstrates that the ES-NC is suitable for the identification of cancerous cells from healthy cells, indicating a promising tool for early diagnosis and prediction of cancer.

R3-MYB transcription factor LcMYBx from Litchi chinensis negatively regulates anthocyanin biosynthesis by ectopic expression in tobacco.

Anthocyanin accumulation is one of the remarkable physiological changes during fruit ripening. In plants, anthocyanin synthesis is regulated by MYB activators, but the MYB repressors has been recognized recently. Here, we isolated a repressor of anthocyanin synthesis, LcMYBx, from Litchi chinensis Sonn. LcMYBx encoded a typical R3-MYB protein and contained a conserved [D/E]Lx2[R/K]x3Lx6Lx3R motif for interacting with bHLH proteins. Overexpression of LcMYBx in tobacco suppressed anthocyanin accumulation resulting in faded petals from pale-pink to almost white. Gene expression analysis showed the strong down-regulation of endogenous anthocyanin structural and regulatory genes by LcMYBx overexpression. Yeast two-hybrid and bimolecular fluorescence complementation assays indicated that LcMYBx could interact with the transcription factors LcbHLH1 and LcbHLH3. Transient promoter activation assays showed that LcMYBx could inhibit the activation capacity of LcMYB1-LcbHLH3 complex for LcDFR gene. These results suggest that LcMYBx competed with LcMYB1 to LcbHLHs, thus preventing the activation of LcDFR by LcMYB1-LcbHLHs complex and negatively controlling anthocyanin biosynthesis.

Advance of the negative regulation of anthocyanin biosynthesis by MYB transcription factors.

Anthocyanins are secondary metabolites derived from the specific branch of the flavonoid pathway, responsible for red, purple and blue coloration display in the flowers and fruits. The functions of anthocyanins are diverse, including acting as visual signals to pollinators, defense against biotic and abiotic stresses. Thus, anthocyanins have been the most intensely studied secondary metabolite pathway. From model plants to horticultural crops, numerous studies have resulted in the discovery of highly conserved MYB-bHLH-WDR (MBW) transcriptional complex for the regulation of anthocyanin biosynthesis in plants. Recent discoveries have revealed that the anthocyanin biosynthesis pathway is also controlled by MYB repressors. Here we focus on the research progress into the role of MYB repressors in anthocyanin biosynthesis. In particular, we will discuss their functions and relationship to the MBW complex in the control of anthocyanin accumulation. In addition, an integrated regulatory network of anthocyanin biosynthesis controlled by MYB repressors and MBW activation complex is built based on the significant progress.

Three LcABFs are Involved in the Regulation of Chlorophyll Degradation and Anthocyanin Biosynthesis During Fruit Ripening in Litchi chinensis.

During litchi (Litchi chinensis Sonn.) fruit ripening, two major physiological changes, degreening (Chl degradation) and pigmentation (anthocyanin biosynthesis), are visually apparent. However, the specific factor triggering this important transition is still unclear. In the present study, we found that endogenous ABA content increased sharply when Chl breakdown was initiated and the ABA level peaked just before the onset of anthocyanin accumulation, suggesting that ABA plays an important role during litchi fruit pigmentation. We characterized three ABSCISIC ACID RESPONSE ELEMENT-BINDING FACTORs (LcABF1/2/3) belonging to group A of the basic leucine zipper (bZIP) transcription factors previously shown to be involved in ABA signaling under abiotic stress. LcABF1 transcripts increased at the onset of Chl degradation, and the expression of LcABF3 accumulated in parallel with anthocyanin biosynthesis. In addition, dual luciferase and yeast one-hybrid assays indicated that LcABF1/2 recognized ABA-responsive elements in the promoter region of Chl degradation-related genes (PAO and SGR), while LcABF2/3 bound the promoter region of LcMYB1 and anthocyanin biosynthesis-related structural genes. Indeed, Nicotiana benthamiana leaves transiently expressing LcABF1/2 showed a senescence phenomenon with Chl degradation, and LcABF3 overexpression increased the accumulation of anthocyanin via activation of LcMYB1, which is the key determinant of anthocyanin biosynthesis. These data indicate that LcABF1/2/3 are important transcriptional regulators of ABA-dependent litchi fruit ripening involved in both Chl degradation and anthocyanin biosynthesis.

Identification and expression profile analysis of the sucrose phosphate synthase gene family in Litchi chinensis Sonn.

Sucrose phosphate synthase (SPS, EC 2.4.1.14) is a key enzyme that regulates sucrose biosynthesis in plants. SPS is encoded by different gene families which display differential expression patterns and functional divergence. Genome-wide identification and expression analyses of SPS gene families have been performed in Arabidopsis, rice, and sugarcane, but a comprehensive analysis of the SPS gene family in Litchi chinensis Sonn. has not yet been reported. In the current study, four SPS gene (LcSPS1, LcSPS2, LcSPS3, and LcSPS4) were isolated from litchi. The genomic organization analysis indicated the four litchi SPS genes have very similar exon-intron structures. Phylogenetic tree showed LcSPS1-4 were grouped into different SPS families (LcSPS1 and LcSPS2 in A family, LcSPS3 in B family, and LcSPS4 in C family). LcSPS1 and LcSPS4 were strongly expressed in the flowers, while LcSPS3 most expressed in mature leaves. RT-qPCR results showed that LcSPS genes expressed differentially during aril development between cultivars with different hexose/sucrose ratios. A higher level of expression of LcSPS genes was detected in Wuheli, which accumulates higher sucrose in the aril at mature. The tissue- and developmental stage-specific expression of LcSPS1-4 genes uncovered in this study increase our understanding of the important roles played by these genes in litchi fruits.