Lifeng Jin

Colorimetric detection of Hg2+ and Pb2+ based on peroxidase-like activity of graphene oxide–gold nanohybrids

In recent years, considerable efforts have been devoted to the construction of efficient enzyme mimetics, which have significant advantages of simple synthesis and good stability. In this paper, we prepared novel graphene oxide–gold (GO–AuNP) nanohybrids that exhibited fascinating peroxidase-like activity and could catalyze the oxidation of the peroxidase substrate 3,3,5,5-tetramethylbenzidine (TMB) by H2O2, while simultaneously developing a blue color in an aqueous solution. Furthermore, single-stranded DNA (ssDNA) were able to resist the salt-induced aggregation of GO–AuNP nanohybrids, whereas double-stranded DNA (dsDNA) did not inhibit salt-induced GO–AuNP nanohybrids aggregation. Based on these unique properties, we developed a label-free colorimetric method for the detection of Hg2+ and Pb2+. In the presence of Hg2+ or Pb2+, ssDNA formed a hairpin-like or a quadruplex structure, and these conformational changes facilitated the salt-induced aggregation of GO–AuNP nanohybrids, leaving behind a transparent supernatant after centrifugation. After incubation with TMB and H2O2, the colorimetric signal of the centrifugal supernatant was significantly decreased compared to that in the absence of Hg2+ or Pb2+. The response to Hg2+ and Pb2+ were in the range of 0–50 μM. Finally, this simple and sensitive sensor could be successfully applied for the detection of Hg2+ and Pb2+ in river water.

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 β-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 β-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.

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

BackgroundDrought 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).ResultsIn 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.ConclusionsThis 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.

Graphene Oxide-Based Homogeneous Fluorescence Sensor for Multiplex Determination of Various Targets by a Multifunctional Aptamer

A sensitive and selective fluorescent aptasensor for the determination of metal ions, small molecules, sequence-specific DNA, and proteins was fabricated. The analytical approach was based on target-induced conformational changes of the probe and the self-assembled probe-graphene oxide architecture. The probe employed fluorescence “on/off” switching in a single step in solution. This approach was simple to prepare and had little background with good sensitivity and rapid response. In the absence of targets, the probe adsorbed on the surface of graphene oxide through π–π stacking and quenched fluorescence of the probe. Upon the addition of analyte, the random coil of the probe bonded to the targets, reducing the interaction between the probe and graphene oxide, which disrupted the energy transfer from the probe to graphene oxide and therefore increased the fluorescence. This approach was employed for the determination of Ag+, Hg2+, cysteine, sequence-specific DNA, and thrombin. The results demonstrated that the probe-graphene oxide architecture was an excellent and versatile platform for the determination of multiple analytes.

Cloning of the Lycopene β-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 β cyclase (β-LCY) functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a β-LCY gene from Nicotiana tabacum, designated as Ntβ-LCY1, was cloned and functionally characterized. Robust expression of Ntβ-LCY1 was found in leaves, and Ntβ-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 Ntβ-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 Ntβ-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 Ntβ-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum.

Downregulation of the lycopene ε-cyclase gene confers tolerance to salt and drought stress in Nicotiana tabacum

Lycopene ε-cyclase (ε-LCY) functions at a branch point of the carotenoid biosynthesis pathway and modulates the ratio of lutein to the β-carotenoids. RNA interference (RNAi) and overexpression (OE) of Ntε-LCY were used to evaluate the physiological roles of ε-LCY in Nicotiana tobacum. In leaves, strong accumulation of β-branch carotenoids and high expression of carotenoid biosynthesis genes resulted from suppression of Ntε-LCY expression. RNAi plants showed enhanced salt and drought tolerance, while overexpression of the Ntε-LCY gene weakened tolerance to salt and drought stress, as compared to control. Further analysis revealed that RNAi plants exhibited less water loss and had lower reactive oxygen species levels than did WT plants after both the salt and drought treatments. Further, higher levels of ABA accumulated in the RNAi lines than in the WT lines under stress conditions. These results suggest that reduced Ntε-LCY expression can improve drought and salinity tolerance in Nicotiana tabacum by enhancing their ROS scavenging ability.

Genome-wide identification and characterization of an amino acid permease gene family in Nicotiana tabacum

The amino acid permease (AAP) protein family is an important class of amino acid transporter. However, little information about this gene family is available in tobacco. Here, a total number of 15, 7, and 6 full-length putative AAP genes have been identified in the genomes of Nicotiana tabacum and progenitors of modern tobacco, Nicotiana sylvestris and Nicotiana tomentosiformis, respectively. We performed a multiple analyses of the AAP gene family in tobacco and reported data including phylogenetic relationships, gene structures, predicted protein structures and information about conserved motifs. These tobacco AAP family members shared high levels of similarity in their nucleotide and amino acid sequences. The gene structures of the NtAAP loci were all separated by introns. Phylogenetic analyses indicated that these AAP genes were clustered into three groups. We also performed expression profiling analyses of the NtAAP genes in various tobacco tissues/organs using quantitative PCR. The differential expression patterns of the NtAAP genes suggested that the family members might have different biological functions in tobacco plants. To study the function of NtAAP2-2, its coding sequence was cloned and overexpression/RNA interference vectors were constructed and these vectors were transformed into tobacco. Compared with wild-type tobacco, the contents of Glu, Gln, Asp and Asn were found to change in the transgenic tobacco leaves, indicating that NtAAP2-2 was involved in the transportation of Glu, Gln, Asp and Asn in tobacco. Our results can serve as fundamental research for future evolutionary and functional characterization studies of the AAP genes in tobacco.

Three duplication events and variable molecular evolution characteristics involved in multiple GGPS genes of six Solanaceae species.

1China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute, Zhengzhou, Henan 450001, People’s Republic of China 2Staff Development Institute of CNTC, Zhengzhou, Henan 450008, People’s Republic of China 3Mudanjiang Tobacco Research Institute, Mudanjiang, Heilongjiang 157001, People’s Republic of China 4Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, Hainan 571737, People’s Republic of China

Spinacia oleracea proteins with antiviral activity against tobacco mosaic virus

Tobacco mosaic virus (TMV) is a devastating microorganism with a global distribution and a wide host range. Protein extracts isolated from Spinacia oleracea (spinach) were examined with bioassay-guide and tested for the resistance to TMV in the Nicotiana glutinosa leaves by local lesion assay. The results show that the crude extract and the fraction of 0 to 40% ammonium sulfate precipitation could protect tobacco from TMV infection. An active fraction was further purified with cation-exchange chromatography (SP-sepharose HP) and activity screening. The inhibitory rate for TMV in vitro was up to 94.35% with 50 µg/mL of the active fraction. In addition, this active fraction could function against TMV at the infection sites on leaves of N. glutinosa . However, it had only a little inhibitory effect on TMV replication and the mechanism under the TMV-inhibitory activity of protein extract from spinach still need further investigation.

Identification and analysis of the chloride channel gene family members in tobacco (Nicotiana tabacum).

The chloride channel (CLC) protein family, which includes both chloride (Cl-) channels and chloride/proton (Cl-/H+) antiporters, is present in all domains of life, from prokaryotes to eukaryotes. However, there are no reported studies about this gene family in tobacco, an economically important global crop plant. In this study, we identified seventeen CLC genes in the genome of Nicotiana tabacum. A multiple sequence alignment showed that all of the predicted proteins shared a high sequence similarity and had a highly conserved GKxGPxxH motif. A gene structure analysis revealed that the NtCLC genes had highly divergent intron-exon patterns. A phylogenetic and conserved motif analysis revealed that the NtCLC family was divided into two clades, in a manner similar to other plants. We also evaluated the expression patterns of these NtCLC genes in different tissues and in plants treated with salt stress. The NtCLC genes had highly variable expression patterns, for example, the largely stem- and bud-specific expression patterns of NtCLC6 and NtCLC8, respectively. Salt stress treatment (300 mM NaCl) induced the expression of NtCLC2, NtCLC3, and NtCLC12, suggesting that these genes might play a role in tobacco responses to salt stress. Furthermore, the concentration of Cl- in the NtCLC2- and NtCLC13-silenced plants showed an obvious lower and higher level, respectively, than the control plants. Thus, we indicated that NtCLC2 or NtCLC13 might play an important role in chloride transport or metabolism in tobacco. Together, these findings establish an empirical foundation for the further functional characterization of the NtCLC genes in tobacco.