Genome-wide analysis of long noncoding RNAs in response to salt stress in Nicotiana tabacum.

BACKGROUND: Long noncoding RNAs (lncRNAs) have been shown to play important roles in the response of plants to various abiotic stresses, including drought, heat and salt stress. However, the identification and characterization of genome-wide salt-responsive lncRNAs in tobacco (Nicotiana tabacum L.) have been limited. Therefore, this study aimed to identify tobacco lncRNAs in roots and leaves in response to different durations of salt stress treatment. RESULTS: A total of 5,831 lncRNAs were discovered, with 2,428 classified as differentially expressed lncRNAs (DElncRNAs) in response to salt stress. Among these, only 214 DElncRNAs were shared between the 2,147 DElncRNAs in roots and the 495 DElncRNAs in leaves. KEGG pathway enrichment analysis revealed that these DElncRNAs were primarily associated with pathways involved in starch and sucrose metabolism in roots and cysteine and methionine metabolism pathway in leaves. Furthermore, weighted gene co-expression network analysis (WGCNA) identified 15 co-expression modules, with four modules strongly linked to salt stress across different treatment durations (MEsalmon, MElightgreen, MEgreenyellow and MEdarkred). Additionally, an lncRNA-miRNA-mRNA network was constructed, incorporating several known salt-associated miRNAs such as miR156, miR169 and miR396. CONCLUSIONS: This study enhances our understanding of the role of lncRNAs in the response of tobacco to salt stress. It provides valuable information on co-expression networks of lncRNA and mRNAs, as well as networks of lncRNAs-miRNAs-mRNAs. These findings identify important candidate lncRNAs that warrant further investigation in the study of plant-environment interactions.

Functional characterization and comparison of lycopene epsilon-cyclase genes in Nicotiana tabacum.

BACKGROUND: Lycopene epsilon-cyclase (ε-LCY) is a key enzyme in the carotenoid biosynthetic pathway (CBP) of higher plants. In previous work, we cloned two Ntε-LCY genes from allotetraploid tobacco (Nicotiana tabacum), Ntε-LCY2 and Ntε-LCY1, and demonstrated the overall effect of Ntε-LCY genes on carotenoid biosynthesis and stress resistance. However, their genetic and functional characteristics require further research in polyploid plants. RESULTS: Here, we used CRISPR/Cas9 to obtain Ntε-LCY2 and Ntε-LCY1 mutants in allotetraploid N.tabacum K326. Ntε-LCY2 and Ntε-LCY1 had similar promoter cis-acting elements, including light-responsive elements. The Ntε-LCY genes were expressed in roots, stems, leaves, flowers, and young fruit, and their highest expression levels were found in leaves. Ntε-LCY2 and Ntε-LCY1 genes responded differently to normal light and high light stress. Both the Ntε-LCY2 and the Ntε-LCY1 mutants had a more rapid leaf growth rate, especially ntε-lcy2-1. The expression levels of CBP genes were increased in the ntε-lcy mutants, and their total carotenoid content was higher. Under both normal light and high light stress, the ntε-lcy mutants had higher photosynthetic capacities and heat dissipation levels than the wild type, and this was especially true of ntε-lcy2-1. The reactive oxygen species content was lower in leaves of the ntε-lcy mutants. CONCLUSION: In summary, the expression patterns and biological functions of the Ntε-LCY genes Ntε-LCY1 and Ntε-LCY2 differed in several respects. The mutation of Ntε-LCY2 was associated with a greater increase in the content of chlorophyll and various carotenoid components, and it enhanced the stress resistance of tobacco plants under high light.

Characterization and functional analysis of phytoene synthase gene family in tobacco.

BACKGROUND: Carotenoids play important roles in photosynthesis, hormone signaling, and secondary metabolism. Phytoene synthase (PSY) catalyzes the first step of the carotenoid biosynthetic pathway. In this study, we aimed to characterize the PSY genes in tobacco and analyze their function. RESULTS: In this study, we identified three groups of PSY genes, namely PSY1, PSY2, and PSY3, in four Nicotiana species; phylogenetic analysis indicated that these genes shared a high similarity with those in tomato but not with those in monocots such as rice and maize. The expression levels of PSY1 and PSY2 were observed to be highest in leaves compared to other tissues, and they could be elevated by treatment with certain phytohormones and exposure to strong light. No PSY3 expression was detected under these conditions. We constructed virus-induced PSY1 and PSY2 silencing in tobacco and found that the newly emerged leaves in these plants were characterized by severe bleaching and markedly decreased carotenoid and chlorophyll content. Thylakoid membrane protein complex levels in the gene-silenced plants were also less than those in the control plants. The chlorophyll fluorescence parameters such as Fv/Fm, ΦPSII, qP, and NPQ, which reflect photosynthetic system activities, of the gene-silenced plants were also significantly decreased. We further performed RNA-Seq and metabonomics analysis between gene-silenced tobacco and control plants. RNA-Seq results showed that abiotic stress, isoprenoid compounds, and amino acid catabolic processes were upregulated, whereas the biosynthesis of cell wall components was downregulated. Metabolic analysis results were consistent with the RNA-Seq. We also found the downstream genes in carotenoid biosynthesis pathways were upregulated, and putative transcription factors that regulate carotenoid biosynthesis were identified. CONCLUSIONS: Our results suggest that PSY can regulate carotenoid contents not only by controlling the first biosynthesis step but also by exerting effects on the expression of downstream genes, which would thereby affect photosynthetic activity. Meanwhile, PSY may affect other processes such as amino acid catabolism and cell wall organization. The information we report here may aid further research on PSY genes and carotenoid biosynthesis.

Reconstruction of the full-length transcriptome of cigar tobacco without a reference genome and characterization of anion channel/transporter transcripts.

BACKGROUND: Cigar wrapper leaves are the most important raw material of cigars. Studying the genomic information of cigar tobacco is conducive to improving cigar quality from the perspective of genetic breeding. However, no reference genome or full-length transcripts at the genome-wide scale have been reported for cigar tobacco. In particular, anion channels/transporters are of high interest for their potential application in regulating the chloride content of cigar tobacco growing on coastal lands, which usually results in relatively high Cl- accumulation, which is unfavorable. Here, the PacBio platform and NGS technology were combined to generate a full-length transcriptome of cigar tobacco used for cigar wrappers. RESULTS: High-quality RNA isolated from the roots, leaves and stems of cigar tobacco were subjected to both the PacBio platform and NGS. From PacBio, a total of 11,652,432 subreads (19-Gb) were generated, with an average read length of 1,608 bp. After corrections were performed in conjunction with the NGS reads, we ultimately identified 1,695,064 open reading frames including 21,486 full-length ORFs and 7,342 genes encoding transcription factors from 55 TF families, together with 2,230 genes encoding long non-coding RNAs. Members of gene families related to anion channels/transporters, including members of the SLAC and CLC families, were identified and characterized. CONCLUSIONS: The full-length transcriptome of cigar tobacco was obtained, annotated, and analyzed, providing a valuable genetic resource for future studies in cigar tobacco.

Using virtual standardized patients to accurately assess information gathering skills in medical students.

Introduction: Practicing a medical history using standardized patients is an essential component of medical school curricula. Recent advances in technology now allow for newer approaches for practicing and assessing communication skills. We describe herein a virtual standardized patient (VSP) system that allows students to practice their history taking skills and receive immediate feedback. Methods: Our VSPs consist of artificially intelligent, emotionally responsive 3D characters which communicate with students using natural language. The system categorizes the input questions according to specific domains and summarizes the encounter. Automated assessment by the computer was compared to manual assessment by trained raters to assess accuracy of the grading system. Results: Twenty dialogs chosen randomly from 102 total encounters were analyzed by three human and one computer rater. Overall scores calculated by the computer were not different than those provided by the human raters, and overall accuracy of the computer system was 87%, compared with 90% for human raters. Inter-rater reliability was high across 19 of 21 categories. Conclusions: We have developed a virtual standardized patient system that can understand, respond, categorize, and assess student performance in gathering information during a typical medical history, thus enabling students to practice their history-taking skills and receive immediate feedback.

NtRLK5, a novel RLK-like protein kinase from Nitotiana tobacum, positively regulates drought tolerance in transgenic Arabidopsis.

Receptor-like protein kinase (RLKs) plays pivotal roles in plant growth and development as well as stress responses. However, little is known about the function of RLKs in Nitotiana tobacum. In the present study, we present data on NtRLK5, a novel RLK-like gene isolated from Hongda (Nitotiana tobacum L.). Expression profile analysis revealed that NtRLK5 was strongly induced by drought and salt stresses. Transient expression of NtRLK5-GFP fusion protein in protoplast showed that NtRLK5 was localized to plasma membrane. Overexpression of NtRLK5 conferred enhanced drought tolerance in transgenic Arabidopsis plants, which was attributed to not only the lower malondialdehyde (MDA) and H2O2 contents, but also the higher antioxidant enzymes activities. Moreover, the expression of several antioxidation- and stress-related genes was also significantly up-regulated in NtRLK5 transgenic plants under drought condition. Taken together, the results suggest that NtRLK5 functions as a positive regulator in drought tolerance.

Integrated mRNA and microRNA analysis identifies genes and small miRNA molecules associated with transcriptional and post-transcriptional-level responses to both drought stress and re-watering treatment in tobacco.

BACKGROUND: Drought stress is one of the most severe problem limited agricultural productivity worldwide. It has been reported that plants response to drought-stress by sophisticated mechanisms at both transcriptional and post-transcriptional levels. However, the precise molecular mechanisms governing the responses of tobacco leaves to drought stress and water status are not well understood. To identify genes and miRNAs involved in drought-stress responses in tobacco, we performed both mRNA and small RNA sequencing on tobacco leaf samples from the following three treatments: untreated-control (CL), drought stress (DL), and re-watering (WL). RESULTS: In total, we identified 798 differentially expressed genes (DEGs) between the DL and CL (DL vs. CL) treatments and identified 571 DEGs between the WL and DL (WL vs. DL) treatments. Further analysis revealed 443 overlapping DEGs between the DL vs. CL and WL vs. DL comparisons, and, strikingly, all of these genes exhibited opposing expression trends between these two comparisons, strongly suggesting that these overlapping DEGs are somehow involved in the responses of tobacco leaves to drought stress. Functional annotation analysis showed significant up-regulation of genes annotated to be involved in responses to stimulus and stress, (e.g., late embryogenesis abundant proteins and heat-shock proteins) antioxidant defense (e.g., peroxidases and glutathione S-transferases), down regulation of genes related to the cell cycle pathway, and photosynthesis processes. We also found 69 and 56 transcription factors (TFs) among the DEGs in, respectively, the DL vs. CL and the WL vs. DL comparisons. In addition, small RNA sequencing revealed 63 known microRNAs (miRNA) from 32 families and 368 novel miRNA candidates in tobacco. We also found that five known miRNA families (miR398, miR390, miR162, miR166, and miR168) showed differential regulation under drought conditions. Analysis to identify negative correlations between the differentially expressed miRNAs (DEMs) and DEGs revealed 92 mRNA-miRNA interactions between CL and DL plants, and 32 mRNA-miRNA interactions between DL and WL plants. CONCLUSIONS: This study provides a global view of the transcriptional and the post-transcriptional responses of tobacco under drought stress and re-watering conditions. Our results establish an empirical foundation that should prove valuable for further investigations into the molecular mechanisms through which tobacco, and plants more generally, respond to drought stress at multiple molecular genetic levels.

Higher Levels of Magnesium and Lower Levels of Calcium in Whole Blood Are Positively Correlated with the Metabolic Syndrome in a Chinese Population: A Case-Control Study.

Magnesium (Mg) and calcium (Ca) are essential for numerous kinds of metabolisms in human body. To investigate the associations between Mg and Ca and the ratio of Ca to Mg (Ca/Mg) in whole blood with metabolic syndrome (MetS) in a Chinese population, a matched case-control study including 204 MetS patients and 204 healthy controls (aged 48-89) was carried out in 2011. MetS were diagnosed according to the criteria of Chinese Diabetes Society. Controls had no abnormal metabolic components and were matched with cases by age, gender and region. Blood samples were collected in the morning after an overnight fast. Whole blood Mg and Ca were determined by flame atomic absorption spectrometry. Subjects who were male constituted 44.1% of the part of this study. The average age was 64.0 ± 7.18, and the average body mass index was 24.3 ± 3.75. The MetS group showed significantly higher Mg and lower Ca and Ca/Mg as compared with the control group. Comparing with the bottom tertile (T1) of Mg, increased ORs for MetS were found in median tertile (T2) and top tertile (T3) of Mg. For Ca, T2 and T3 were negatively associated with MetS. Inverse relationship was also found between Ca/Mg ratio and MetS. Our findings suggested that increased Mg and decreased Ca and Ca/Mg in whole blood were correlated with MetS in Chinese adults.

Cloning of the Lycopene ß-cyclase Gene in Nicotiana tabacum and Its Overexpression Confers Salt and Drought Tolerance.

Carotenoids are important pigments in plants that play crucial roles in plant growth and in plant responses to environmental stress. Lycopene ß cyclase (ß-LCY) functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a ß-LCY gene from Nicotiana tabacum, designated as Ntß-LCY1, was cloned and functionally characterized. Robust expression of Ntß-LCY1 was found in leaves, and Ntß-LCY1 expression was obviously induced by salt, drought, and exogenous abscisic acid treatments. Strong accumulation of carotenoids and expression of carotenoid biosynthesis genes resulted from Ntß-LCY1 overexpression. Additionally, compared to wild-type plants, transgenic plants with overexpression showed enhanced tolerance to salt and drought stress with higher abscisic acid levels and lower levels of malondialdehyde and reactive oxygen species. Conversely, transgenic RNA interference plants had a clear albino phenotype in leaves, and some plants did not survive beyond the early developmental stages. The suppression of Ntß-LCY1 expression led to lower expression levels of genes in the carotenoid biosynthesis pathway and to reduced accumulation of carotenoids, chlorophyll, and abscisic acid. These results indicate that Ntß-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum.

Aphanamixins A-F, acyclic diterpenoids from the stem bark of Aphanamixis polystachya.

Six new acyclic diterpenoids named Aphanamixins A-F (1-6), together with two known compounds of nemoralisin and nemoralisin C, were isolated from the stem bark of Aphanamixis polystachya (WALL) J. N. BARKER. Their structures were established through a comprehensive analysis of NMR spectroscopic data and high resolution mass spectrometric data. The absolute configurations of carbon stereocenters were determined by means of auxiliary chiral α-methoxy-α-(trifluoromethyl)phenylacetic acid (MTPA) derivatives and circular dichroism (CD), respectively. All the new isolates were tested for their antiproliferative activity against HepG2, AGS, MCF-7, and A-549 cancer cell lines and they exhibited weak cytotoxicities (IC50>10 µM). Moreover, we highlighted that the six new diterpenoids characterized by acyclic skeleton was rarely seen in nature.

Molecular cloning and functional characterization of the lycopene ε-cyclase gene via virus-induced gene silencing and its expression pattern in Nicotiana tabacum.

Lycopene ε-cyclase (ε-LCY) is a key enzyme that catalyzes the synthesis of α-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding ε-LCY (designated Ntε-LCY1 and Ntε-LCY2) were cloned from Nicotiana tabacum. Ntε-LCY1 and Ntε-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Ntε-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Ntε-LCY1-GFP fusion protein demonstrated that mature Ntε-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Ntε-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV)-mediated silencing of ε-LCY in Nicotiana benthamiana resulted in an increase of ß-branch carotenoids and a reduction in the levels of α-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of ß-OHase in the TRV-ε-lcy line. Suppression of ε-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS) plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of ε-LCY in plant carotenoid biosynthesis and suggest a role for ε-LCY in positively modulating low temperature stress responses.

Investigation of the relationship between the metabolic profile of tobacco leaves in different planting regions and climate factors using a pseudotargeted method based on gas chromatography/mass spectrometry.

An improved pseudotargeted method using gas chromatography/mass spectrometry (GC/MS) was developed to investigate the metabolic profile of tobacco leaves from three planting regions (Yunnan, Guizhou, and Henan provinces). The analytical characteristics of the method with regard to reproducibility, precision, linearity, and stability were satisfactory for metabolic profiling study. Partial least-squares-discriminant analysis and hierarchical cluster analysis demonstrated that the metabolic profiles of tobacco from the Yunnan and Guizhou regions were different from that from the Henan province. The amino acid (e.g., phenylalanine, leucine, and tyrosine) and carbohydrate (e.g., fructose, trehalose, and sucrose) contents were the highest in Henan tobacco. The highest contents of organic acids (e.g., isocitrate, citrate, and fumarate) of the TCA cycle and antioxidants (e.g., quinate, chlorogenic acid, and ascorbate) were found in Guizhou tobacco. The correlation coefficients between metabolite content and climate factors (rainfall, sunshine, and temperature) demonstrated that drought facilitated the accumulation of sugars and amino acids. The content of TCA cycle intermediates could be influenced by multiple climate factors. This study demonstrates that the pseudotargeted method with GC/MS is suitable for the investigation of the metabolic profiling of tobacco leaves and the assessment of differential metabolite levels related to the growing regions.

Modulation of lipogenesis and glucose consumption in HepG2 cells and C2C12 myotubes by sophoricoside.

Sophoricoside, an isoflavone glycoside isolated from Sophora japonica (Leguminosae), has been widely reported as an immunomodulator. In this study, the effects of sophoricoside on lipogenesis and glucose consumption in HepG2 cells and C2C12 myotubes were investigated. Treatment with sophoricoside at concentrations of 1-10 µM inhibited lipid accumulation in HepG2 cells in a dose-dependent manner. At the same concentration range, no effect on cell viability was observed in the MTT assay. Inhibition of lipogenesis was associated with the downregulation of SREBP-1a, SREBP-1c, SREBP-2 and their downstream target genes (FAS, ACC, HMGR) as revealed by realtime quantitative PCR. The lipid-lowering effect was mediated via the phosphorylation of AMPK. Further investigation of the activities of this isoflavone showed that sophoricoside has the capability to increase glucose uptake by C2C12 myotubes. It also effectively inhibited the activities of α-glucosidase and α-amylase in vitro and remarkably lowered postprandial hyperglycaemia in starch-loaded C57BL6/J mice. These results suggest that sophoricoside is an effective regulator of lipogenesis and glucose consumption and may find utility in the treatment of obesity and type 2 diabetes.

Dynamic Augmentation Data Selection for Few-shot Text Classification.

Data augmentation has been a popular method for fine-tuning pre-trained language models to increase model robustness and performance. With augmentation data coming from modifying gold train data (in-sample augmentation) or being harvested from general domain unlabeled data (out-of-sample augmentation), the quality of such data is the key to successful fine-tuning. In this paper, we propose a dynamic data selection method to select effective augmentation data from different augmentation sources according to the model's learning stage, by identifying a set of augmentation samples that optimally facilitates the learning process of the most current model. The method firstly filters out augmentation samples with noisy pseudo labels through a curriculum learning strategy, then estimates the effectiveness of reserved augmentation data by its influence scores on the current model at every update, allowing the data selection process tightly tailored to model parameters. And the two-stage augmentation strategy considers in-sample augmentation and out-of-sample augmentation in different learning stages. Experiments with both kinds of augmentation data on a variety of sentence classification tasks show that our method outperforms strong baselines, proving the effectiveness of our method. Analysis confirms the dynamic nature of the data effectiveness and the importance of model learning stages in utilization of augmentation data.

Rational design of geranylgeranyl diphosphate synthase enhances carotenoid production and improves photosynthetic efficiency in Nicotiana tabacum.

Restricted genetic diversity can supply only a limited number of elite genes for modern plant cultivation and transgenesis. In this study, we demonstrate that rational design enables the engineering of geranylgeranyl diphosphate synthase (NtGGPPS), an enzyme of the methylerythritol phosphate pathway (MEP) in the model plant Nicotiana tabacum. As the crucial bottleneck in carotenoid biosynthesis, NtGGPPS1 interacts with phytoene synthase (NtPSY1) to channel GGPP into the production of carotenoids. Loss of this enzyme in the ntggpps1 mutant leads to decreased carotenoid accumulation. With the aim of enhancing NtGGPPS1 activity, we undertook structure-guided rational redesign of its substrate binding pocket in combination with sequence alignment. The activity of the designed NtGGPPS1 (a pentuple mutant of five sites V154A/I161L/F218Y/I209S/V233E, d-NtGGPPS1) was measured by a high-throughput colorimetric assay. d-NtGGPPS1 exhibited significantly higher conversion of IPP and each co-substrate (DMAPP ~1995.5-fold, GPP ~25.9-fold, and FPP ~16.7-fold) for GGPP synthesis compared with wild-type NtGGPPS1. Importantly, the transient and stable expression of d-NtGGPPS1 in the ntggpps1 mutant increased carotenoid levels in leaves, improved photosynthetic efficiency, and increased biomass relative to NtGGPPS1. These findings provide a firm basis for the engineering of GGPPS and will facilitate the development of quality and yield traits. Our results open the door for the structure-guided rational design of elite genes in higher plants.

Genome-wide identification and characterization of NPF family reveals NtNPF6.13 involving in salt stress in Nicotiana tabacum.

Proteins of the Nitrate Transporter 1/Peptide Transporter (NPF) family transport a diverse variety of substrates, such as nitrate, peptides, hormones and chloride. In this study, a systematic analysis of the tobacco (Nicotiana tabacum) NPF family was performed in the cultivated 'K326'. In total, 143 NtNPF genes were identified and phylogenetically classified into eight subfamilies, NPF1 to NPF8, based on the classification of NPF families in other plant species. The chromosomal locations and structures of the NtNPF genes were analyzed. The expression profiles of NtNPF genes under NaCl stress were analyzed to screen the possible NPF genes involving in chloride regulation in tobacco. Most NtNPF6 genes responded to salt stress in the roots and leaves. The expression of NtNPF6.13 was significantly down-regulated after salt stress for 12h. The chloride content was reduced in the roots of ntnpf6.13 mutant. These findings support the participation of NtNPF6.13 in chloride uptake. Several other NtNPF genes that play potential roles in chloride metabolism of tobacco require further study.

A Fast and Highly Selective Nitrite Sensor Based on Interdigital Electrodes Modified With Nanogold Film and Chrome-Black T.

Nitrite is a toxic substance, when excessive nitrite enters the human body, it will be seriously harmful to human. At present, the detection methods of nitrite are complicated to operate and require expensive detection instruments. Therefore, an effective, fast and highly selective nanogold film interdigital electrode sensors that can detect nitrite easily and quickly is developed in the work. Firstly, the variation of the sensitivity of nanogold film nitrite sensors with concentrations (1 mol/L, 10-1 mol/L, 10-2 mol/L, 10-3 mol/L, 10-4 mol/L, and 10-5 mol/L) was measured by experiments. Then, Chrome-black T was modified to the surface of the nanogold film interdigital electrodes by electrochemical polymerization, and the film of chrome-black T had affinity for nitrite ions, so nitrite ions were enriched on the sensor surface. The change law of the impedance signal of the modified nanogold film nitrite sensors after being added to different concentrations of sodium nitrite solution were also concluded. The study demonstrates that the larger the concentration of sodium nitrite solution is added to the modified interdigital electrodes, the smaller impedance and resistance of the modified interdigital electrodes are reflected. Finally, specificity of the modified interdigital electrode sensors has been demonstrated. The novel interdigital electrode sensors can detect the concentration of nitrite solution conveniently and quickly with only 30 s. Therefore, the prospect of applying the novel nanogold film interdigital electrode sensors to the detection of nitrite in blood, body fluid, food and drinking water is promising.

Study on AgInZnS-Graphene Oxide Non-toxic Quantum Dots for Biomedical Sensing.

In recent years, non-toxic quantum dot has caught the attention of biomedical fields. However, the inherent cytotoxicity of QDs makes its biomedical application painful, and is a major drawback of this method. In this paper, a non-toxic and water-soluble quantum dot AgInZnS-GO using graphene oxide was synthesized. A simple model of state complex was also established, which is produced through a combination of quantum dots and protein. The interaction between AIZS-GO QDs and human serum albumin (HSA) has significant meaning in vivo biological application. Herein, the binding of AIZS-GO QDs and HSA were researched using fluorescence spectra, Uv-visible absorption spectra, FT-IR spectra, and circular dichroism (CD) spectra. The results of fluorescence spectra demonstrate that AIZS-GO QDs have an obvious fluorescence quenching effect on HSA. The quenching mechanism is static quenching, which implies that some type of complex was produced by the binding of QDs and HSA. These results were further proved by Uv-visible absorption spectroscopy. The Stern-Volmer quenching constant Ksv at various temperatures (298 K, 303 K, 308 K) were acquired from analyzing Stern-Volmer plots of the fluorescence quenching information. The Van't Hoff equation could describe the thermodynamic parameters, which demonstrated that the van der Waals and hydrogen bonds had an essential effect on the interaction. FT-IR spectra and CD spectra further indicate that AIZS-GO QDs can alter the structure of HSA. These spectral methods show that the quantum dot can combine well with HSA. The experimental results showed that AgInZn-GO water-soluble quantum dots have good biocompatibility, which can be combined with proteins to form new compounds which have no cytotoxicity and biological practicability. It provides an important basis for the combination of quantum dots and specific proteins as well as fluorescent labeling.

Study on Interaction Between TATA-Box Binding Protein (TBP), TATA-Box and Multiprotein Bridging Factor 1(MBF1) in Beauveria bassiana by Graphene-Based Electrochemical Biosensors.

The regulation of transcription level is an important step in gene expression process. Beauveria bassiana is a broad-spectrum insecticidal fungi widely used in the biologic control of arthropod. The regulation of its transcription level is a multilevel complex process. Multiprotein bridging factor 1(MBF1) is a transcriptional co-activator that bridges sequence-specific activators and the TATA-box binding protein(TBP), Little is known about the interaction between MBF1, TBP, and TBP binding to DNA(TATA-sequences)in filamentous fungi of Beauveria bassiana, The binding of TBP to TATA-box and TBP to MBF1 was investigated via electrochemical biosensor. Graphene oxide has an electronic mobility that is unattainable for any metal, so it will be highly sensitive as a test electrode. Hence, we developed a simple, sensitive and specific sensor based on an TBP probe and graphene oxide that successfully detected the interaction of TBP and TATA-box or MBF1. From the electrochemical impedance spectroscopy (EIS), we find that the radius will increase when adding TATA-box or MBF1 buffer to the modified TBP protein electrode. When adding no TATA-box or no MBF1, the radius is relatively unchanged. The interaction between TBP and TATA-box or MBF1 was proved based on the results. These data confirmed the specificity of the interactions, (1) our developed graphene-based electrochemical biosensor can be used for monitoring the interaction between TBP and TATA-box or MBF1, (2) TBP can bind to TATA-box, (3) TBP can bind to MBF1, and (4) TBP mediates the interactions of MBF1 to DNA. Therefore, this work provided a label-free, low-cost and simple detection method for the complex process of eukaryotic gene transcription regulation.

Integrating transcriptome and metabolome reveals molecular networks involved in genetic and environmental variation in tobacco.

Tobacco (Nicotiana tabacum) is one of the most widely cultivated commercial non-food crops with significant social and economic impacts. Here we profiled transcriptome and metabolome from 54 tobacco samples (2-3 replicates; n = 151 in total) collected from three varieties (i.e. genetic factor), three locations (i.e. environmental factor), and six developmental stages (i.e. developmental process). We identified 3,405 differentially expressed (DE) genes (DEGs) and 371 DE metabolites, respectively. We used quantitative real-time PCR to validate 20 DEGs, and confirmed 18/20 (90%) DEGs between three locations and 16/20 (80%) with the same trend across developmental stages. We then constructed nine co-expression gene modules and four co-expression metabolite modules , and defined seven de novo regulatory networks, including nicotine- and carotenoid-related regulatory networks. A novel two-way Pearson correlation approach was further proposed to integrate co-expression gene and metabolite modules to identify joint gene-metabolite relations. Finally, we further integrated DE and network results to prioritize genes by its functional importance and identified a top-ranked novel gene, LOC107773232, as a potential regulator involved in the carotenoid metabolism pathway. Thus, the results and systems-biology approaches provide a new avenue to understand the molecular mechanisms underlying complex genetic and environmental perturbations in tobacco.