Regulation of autophagy and lipid accumulation under phosphate limitation in Rhodotorula toruloides.

Background: It is known that autophagy is essential for cell survival under stress conditions. Inorganic phosphate (Pi) is an essential nutrient for cell growth and Pi-limitation can trigger autophagy and lipid accumulation in oleaginous yeasts, yet protein (de)-phosphorylation and related signaling events in response to Pi limitation and the molecular basis linking Pi-limitation to autophagy and lipid accumulation remain elusive. Results: Here, we compared the proteome and phosphoproteome of Rhodotorula toruloides CGMCC 2.1389 under Pi-limitation and Pi-repletion. In total, proteome analysis identified 3,556 proteins and the phosphoproteome analysis identified 1,649 phosphoproteins contained 5,659 phosphosites including 4,499 pSer, 978 pThr, and 182 pTyr. We found Pi-starvation-induced autophagy was regulated by autophagy-related proteins, but not the PHO pathway. When ATG9 was knocked down, the engineered strains produced significantly less lipids under Pi-limitation, suggesting that autophagy required Atg9 in R. toruloides and that was conducive to lipid accumulation. Conclusion: Our results provide new insights into autophagy regulation under Pi-limitation and lipid accumulation in oleaginous yeast, which should be valuable to guide further mechanistic study of oleaginicity and genetic engineering for advanced lipid producing cell factory.

Single-Molecule Real-Time and Illumina Sequencing to Analyze Transcriptional Regulation of Flavonoid Synthesis in Blueberry.

Blueberries (Vaccinium corymbosum) contain large amounts of flavonoids, which play important roles in the plant's ability to resist stress and can also have beneficial effects on human health when the fruits are eaten. However, the molecular mechanisms that regulate flavonoid synthesis in blueberries are still unclear. In this study, we combined two different transcriptome sequencing platforms, single-molecule real-time (SMRT) and Illumina sequencing, to elucidate the flavonoid synthetic pathways in blueberries. We analyzed transcript quantity, length, and the number of annotated genes. We mined genes associated with flavonoid synthesis (such as anthocyanins, flavonols, and proanthocyanidins) and employed fluorescence quantitative PCR to analyze the expression of these genes and their correlation with flavonoid synthesis. We discovered one R2R3 MYB transcription factor from the sequencing library, VcMYB1, that can positively regulate anthocyanin synthesis in blueberries. VcMYB1 is mainly expressed in colored (mature) fruits. Experiments showed that overexpression and transient expression of VcMYB1 promoted anthocyanin synthesis in Arabidopsis, tobacco (Nicotiana benthamiana) plants and green blueberry fruits. Yeast one-hybrid (Y1H) assay, electrophoretic mobility shift assay, and transient expression experiments showed that VcMYB1 binds to the MYB binding site on the promoter of the structural gene for anthocyanin synthesis, VcMYB1 to positively regulate the transcription of VcDFR, thereby promoting anthocyanin synthesis. We also performed an in-depth investigation of transcriptional regulation of anthocyanin synthesis. This study provides background information and data for studying the synthetic pathways of flavonoids and other secondary metabolites in blueberries.

[The Characteristic Research of ·OH Induced by Water on an Argon Plasma Jet].

·OH plays a crucial role in many fields, having aroused wide public concern in the world. Atmospheric Pressure Plasma Jet, which can be achieved by portable device due to working without the vacuum environment, has the advantages of high concentration of reactive species, high electron temperature and low gas temperature. It has become an important research topic in the field of gas discharge with a strong prospect. Especially, how to induce plasma jet to produce ·OH has become a new hotpot in the field of low-temperature plasma. It has been reported that mass ·OH can be induced successfully when water vapor is added to the working gas, but it will be unstable when the concentrate of water reaches a certain degree. Thus, a device of argon plasma jet with a Ring-to-Ring Electrode Configuration has been designed to interact with water in the surrounding air to generate ·OH under atmospheric pressure. In order to increase the production of ·OH, ultrasonic atomizing device is introduced to promote water concentration around the plasma plume. The generating rule of OH(A2J) induced by water has been extensively studied under different voltages and flow rate. ·OH output induced by the plasma has been tested by emission spectrometry, and at the meanwhile, Ar atomic spectral lines at 810.41 and 811.48 nm are also recorded in order to calculate the electron temperature in argon plasma plume. The results show that the water surrounding the plasma plume can be induced to produce ·OH, and OH(A2 ∑+) output increases with the electrode voltage rising from 20 to 28 kV. When the flow rate increases from 100 to 200 L x h(-1), the OH(A2∑+) output increases, but from 200 to 600 L x h(-1), it decreases. The production rules of OH(A2∑+) is the same as that of electron temperature. Therefore, the presumption is proved that ·OH output mainly affected by electron temperature.

[Detection of Lead in Water by Electrolyte Cathode Atmospheric Glow Discharge Emission Spectroscopy].

A device based on electrolyte cathode atmospheric glow discharge atomic emission spectroscopy (ELCAD-AES) has been developed to determine the metal ion Pb in water. The emission intensity of Ph was significantly enhanced with the increase concentration of Pb, and the emission intensity has a linear relationship with concentration while the concentration of Pb in the range of 10-80 mg x L(-1). The effects of discharge current and easily ionizable elements on the emission spectral of Pb were investigated, and the emission intensity reached greatest when the discharge current increased to 70 mA, and the easily ionizable elements generated weak effect on the emission spectral of Pb. The effect of acidification regent on emission spectral of Pb was discussed. It was found that it perform best when acidified with HNO3, and reducing the pH can improve the emission intensity of Pb effectively. The emission intensity of Pb at different region was detected near cathode region, thus obtained the best detection position. Under the optimized experimental parameters, the detection limit of Ph was 0.7 mg x L(-1) and relative standard deviation was 1.7%. The recovery of samples was 95%-106%, result and shows that this method has better accuracy. These results provide an available method for further research of detection trace heavy mental elements in water using ELCAD-AES.

mosR, a novel transcriptional regulator of hypoxia and virulence in Mycobacterium tuberculosis.

Latent tuberculosis represents a high-risk burden for one-third of the world population. Previous analysis of murine tuberculosis identified a novel transcriptional regulator encoded by Rv0348 that could control the establishment of persistent tuberculosis. Disruption of the Rv0348 gene from the genome of the virulent H37Rv strain of Mycobacterium tuberculosis revealed a global impact on the transcriptional profiles of 163 genes, including induction of the mammalian cell entry (mce1) operon and the repression of a significant number of genes involved in hypoxia and starvation responses. Nonetheless, gel shift assays did not reveal direct binding between Rv0348 and a set of regulated promoters, suggesting an indirect regulatory role. However, when expressed in Mycobacterium smegmatis, the Rv0348 transcripts were significantly responsive to different levels of hypoxia and the encoded protein was shown to regulate genes involved in hypoxia [e.g., Rv3130c (tgs1)] and intracellular survival (e.g., mce1), among other genes. Interestingly, the colonization level of the DeltamosR mutant strain was significantly lower than that of the wild-type strain of M. tuberculosis, suggesting its attenuation in the murine model of tuberculosis. Taken together, our analyses indicated that the Rv0348 gene encodes a novel transcriptional factor that regulates several operons involved in mycobacterial survival, especially during hypoxia; hence, we propose that Rv0348 be renamed mosR for regulator of mycobacterial operons of survival.

Cloning and Sequencing of cDNA Encoding the Diazepam Binding Inhibitor (DBI).

The anti-morphine peptide PPC from dog brain has an inhibitory effect on morphine analgesia. It is highly homologous to DBI. The analgesic effect of bovine DBI was measured, showing the potentiation effect on morphine analgesia. In order to study the relationship between PPC and DBI, cDNA library derived from dog liver had been screened using partial gene of rat DBI as a probe. A positive clone was obtained. The entire sequence of the insert of this positive clone provided complete sequence of a peptide which was identified as the DBI peptide. It shows 88.5% homology with pig DBI, 87.4% with bovine, 83.9% with human, 46.5% with yeast, respectively. The sequence of N-terminal 50 amino acid residues except 55th, 58th site and C-terminal 17 amino acids is the same to DBI from dog. PPC has 23 amino acids more than DBI inside.