Photoluminescence (PL) intermittency (or “blinking”) is a unique characteristic of single quantum dot (QD) emission. Here, we report a novel single-molecule detection strategy for the intracellular ...mRNA of interest using the mRNA-induced nonblinking QD dimers as probes. The working principle of the method is that the DNA hybrid of the target DNA (or mRNA) with a biotin-modified ssDNA probe can induce two blinking streptavidin-modified QDs (SAV-QDs) conjugated. The formed QD dimer as a bright spot showed a nonblinking emission property, observed with total inner reflection fluorescence microscopy (TIRFM). In theory, one nonblinking spot indicated a target DNA (or mRNA). The experimental results from single-spot fluorescence trajectory analysis and single-particle brightness analysis based on TIRFM and fluorescence correlation spectroscopy (FCS) techniques verified this dimerization process of QDs or its induced nonblinking emission. Employing a target DNA with the same base sequences to Survivin mRNA as a model, the detection strategy was used to detect the target DNA concentration based on the linear relationship between the percentage of the nonblinking spots and the target DNA concentration. This single-molecule detection strategy was also successfully used for determining Survivin mRNA in a single HeLa cell. The method can simplify the hybridization steps, eliminate self-quenching and photobleaching of fluorophores, and reduce the influence of unspecific binding on the detection.
Kiwifruit is a perennial fruit tree with high nutritional and economic value; however, various pathogen stresses have resulted in reductions in its yield and quality. Pentatricopeptide repeat ...proteins (PPRs), characterized by tandem repetitions of 35 amino acid motifs, play roles in RNA editing, mRNA stability, and splicing. They may also regulate plant development and growth. Nevertheless, the roles of PPRs in plant development and disease resistance remain unclear. In this study, we focused on the roles of PPRs in the fruit development and pathogen stress of kiwifruit and conducted a series of analyses of the PPR gene family in two representative kiwifruit species (Actinidia chinensis (Ach) and Actinidia eriantha (Ace)) with markedly different degrees of disease resistance. A total of 497 and 499 PPRs were identified in Ach and Ace, respectively. All the kiwifruit PPRs could be phylogenetically divided into four subfamilies. There were about 40.68% PPRs predicted to be localized to mitochondria or chloroplasts. A synteny analysis showed that the expansion of the kiwifruit PPRs mainly originated from segmental duplication. Based on RNA-seq data from the fruit over 12 periods of development and maturity, a weighted correlation network analysis suggested that two PPRs, Actinidia20495.t1 and Actinidia15159.t1, may be involved in fruit development and maturation. In addition, we observed different responses with respect to the expression of PPRs and RNA editing between resistant and susceptible kiwifruits following infection with pathogenic bacteria, indicating the regulatory role of PPRs in the stress response via the modulation of RNA editing. The differentially expressed upstream transcription factors of the PPRs were further identified; they may regulate resistance adaption by modulating the expression of the PPRs. Collectively, these results suggest that PPRs play roles in the development and disease resistance of kiwifruit and provide candidate genes for further clarifying the resistance mechanisms in kiwifruits.
Sweet cherry (Prunus avium L.), one of the most appreciated and most important commercial temperate fruits, has high sensory quality and nutritional value. Investigating its metabolic variations ...provides valuable information on the formation of fruit quality. In this study, widely targeted LC-MS/MS based metabolomics was used to identify and quantify metabolic changes during 'Black Pearl' sweet cherry development and ripening. A total of 263 significant differentially expressed metabolites (DEMs) were detected during the four fruit-development stages. Significant differences were observed in the composition and content of compounds in the four stages of cherry development, especially sugars, organic acids, and flavonoids. Moreover, transcriptome analysis provided a molecular basis for metabolic variations during fruit development. A total of 6724 significant differentially expressed genes (DEGs) were identified. Further correlation analysis of major DEMs and DEGs showed that 19 key DEGs were involved in sugar metabolism, 23 key DEGs in organic acid metabolism, and 13 key DEGs in flavonoid metabolism. The upregulated genes involved in the flavonoid pathway probably play an important role in regulating the rapid increase of anthocyanin content during fruit development. These comprehensive analysis data provide a better understanding to improve fruit quality traits based on molecular and metabolic levels.
Leucine-rich repeat receptor-like proteins (
), a major group of receptor-like proteins in plants, have diverse functions in plant physiology, including growth, development, signal transduction, and ...stress responses. Despite their importance, the specific roles of kiwifruit
in response to biotic and abiotic stresses remain poorly understood. In this study, we performed family identification, characterization, transcriptome data analysis, and differential gene expression analysis of kiwifruit
. We identified totals of 101, 164, and 105
in
'Hongyang',
'Huate', and
'Red5', respectively. Synteny analysis revealed that the expansion of kiwifruit
was primarily attributed to segmental duplication events. Based on RNA-seq data from pathogen-infected kiwifruits, we identified specific
genes potentially involved in different stages of pathogen infection. Additionally, we observed the potential involvement of kiwifruit
in abiotic stress responses, with upstream transcription factors possibly regulating their expression. Furthermore, protein interaction network analysis unveiled the participation of kiwifruit LRR-RLP in the regulatory network of abiotic stress responses. These findings highlight the crucial roles of
in mediating both biotic and abiotic stress responses in kiwifruit, offering valuable insights for the breeding of stress-resistant kiwifruit varieties.
Grass carp is an important farmed fish in China that is affected by serious disease, especially hemorrhagic disease caused by grass carp reovirus (GCRV). The mechanism underlying the hemorrhagic ...symptoms in infected fish remains to be elucidated. Although GCRV can be divided into three distinct subtypes, differences in the pathogenesis and host immune responses to the different subtypes are still unclear. The aim of this study was to provide a comprehensive insight into the grass carp response to different GCRV subtypes and to elucidate the mechanism underlying the hemorrhagic symptoms.
Following infection of grass carp, GCRV-I was associated with a long latent period and low mortality (42.5%), while GCRV-II was associated with a short latent period and high mortality (81.4%). The relative copy number of GCRV-I remained consistent or decreased slightly throughout the first 7 days post-infection, whereas a marked increase in GCRV-II high copy number was detected at 5 days post-infection. Transcriptome sequencing revealed 211 differentially expressed genes (DEGs) in Group I (66 up-regulated, 145 down-regulated) and 670 (386 up-regulated, 284 down-regulated) in Group II. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed significant enrichment in the terms or pathways involved in immune responses and correlating with blood or platelets. Most of the DEGs in Group I were also present in Group II, although the expression profiles differed, with most DEGs showing mild changes in Group I, while marked changes were observed in Group II, especially the interferon-related genes. Many of the genes involved in the complement pathway and coagulation cascades were significantly up-regulated at 7 days post-infection in Group II, suggesting activation of these pathways.
GCRV-I is associated with low virulence and a long latent period prior to the induction of a mild host immune response, whereas GCRV-II is associated with high virulence, a short latent period and stimulates a strong and extensive host immune response. The complement and coagulation pathways are significantly activated at 7 days post-infection, leading to the endothelial cell and blood cell damage that result in hemorrhagic symptoms.
The flowering transition which is controlled by a complex and intricate gene regulatory network plays an important role in the reproduction for offspring of plants. It is a challenge to identify the ...critical transition state as well as the genes that control the transition of flower development. With the emergence of massively parallel sequencing, a great number of time-course transcriptome data greatly facilitate the exploration of the developmental phase transition in plants. Although some network-based bioinformatics analyses attempted to identify the genes that control the phase transition, they generally overlooked the dynamics of regulation and resulted in unreliable results. In addition, the results of these methods cannot be self-explained.
In this work, to reveal a critical transition state and identify the transition-specific genes of flower development, we implemented a genome-wide dynamic network analysis on temporal gene expression data in Arabidopsis by dynamic network biomarker (DNB) method. In the analysis, DNB model which can exploit collective fluctuations and correlations of different metabolites at a network level was used to detect the imminent critical transition state or the tipping point. The genes that control the phase transition can be identified by the difference of weighted correlations between the genes interested and the other genes in the global network. To construct the gene regulatory network controlling the flowering transition, we applied NARROMI algorithm which can reduce the noisy, redundant and indirect regulations on the expression data of the transition-specific genes. In the results, the critical transition state detected during the formation of flowers corresponded to the development of flowering on the 7th to 9th day in Arabidopsis. Among of 233 genes identified to be highly fluctuated at the transition state, a high percentage of genes with maximum expression in pollen was detected, and 24 genes were validated to participate in stress reaction process, as well as other floral-related pathways. Composed of three major subnetworks, a gene regulatory network with 150 nodes and 225 edges was found to be highly correlated with flowering transition. The gene ontology (GO) annotation of pathway enrichment analysis revealed that the identified genes are enriched in the catalytic activity, metabolic process and cellular process.
This study provides a novel insight to identify the real causality of the phase transition with genome-wide dynamic network analysis.
Multiple organellar RNA editing factor (MORF) genes play key roles in chloroplast developmental processes by mediating RNA editing of Cytosine-to-Uracil conversion. However, the function of MORF ...genes in peach (Prunus persica), a perennial horticultural crop species of Rosaceae, is still not well known, particularly the resistance to biotic and abiotic stresses that threaten peach yield seriously.
In this study, to reveal the regulatory roles of RNA editing in plant immunity, we implemented genome-wide analysis of peach MORF (PpMORF) genes in response to biotic and abiotic stresses. The chromosomal and subcellular location analysis showed that the identified seven PpMORF genes distributed on three peach chromosomes were mainly localized in the mitochondria and chloroplast. All the PpMORF genes were classified into six groups and one pair of PpMORF genes was tandemly duplicated. Based on the meta-analysis of two types of public RNA-seq data under different treatments (biotic and abiotic stresses), we observed down-regulated expression of PpMORF genes and reduced chloroplast RNA editing, especially the different response of PpMORF2 and PpMORF9 to pathogens infection between resistant and susceptible peach varieties, indicating the roles of MORF genes in stress response by modulating the RNA editing extent in plant immunity. Three upstream transcription factors (MYB3R-1, ZAT10, HSFB3) were identified under both stresses, they may regulate resistance adaption by modulating the PpMORF gene expression.
These results provided the foundation for further analyses of the functions of MORF genes, in particular the roles of RNA editing in plant immunity. In addition, our findings will be conducive to clarifying the resistance mechanisms in peaches and open up avenues for breeding new cultivars with high resistance.
The blinking behavior of single quantum dots (QDs) is an intrinsic drawback for some biological and photoelectric applications that rely on single-dot emission. Some studies demonstrate that the ...blinking behavior of QDs is mainly attributed to nonradiative recombination processes associated with traps at the nanocrystal surface. In this study, we systematically investigated the effects of surface ligand alkylthiols on the blinking of CdSe/CdS QDs prepared in the organic phase and observed that the blinking of CdSe/CdS QDs was significantly dependent on the annealing time and structure and concentration of alkylthiol ligands. In the optimal conditions, we prepared thiol-modified CdSe/CdS QDs with a “nonblinking” fraction up to 83%. The mechanism of the blinking suppression was mainly attributed to modifying surface traps of QDs with alkylthiols. In this modification, the decomposition of alkylthiols can slowly release activated “S” under high temperature. Then the activated S can bind to surface traps of QDs, which induces a secondary growth of the core/shell QDs coupled with surface reconstruction and the efficient suppression of the blinking. The method described here can be used to suppress the blinking of other QDs.
Aroma is an important organoleptic quality for fruit and has a large influence on consumer preference. Kiwifruit esters undergo rapid and substantial changes contributing to the flavor during fruit ...ripening. Part of enzymes and their coding genes have been indicated potential candidates for flavor-related esters synthesis. However, there still exist obvious gaps in the biosynthetic pathways of esters and the mechanisms regulating ester biosynthesis in kiwifruit remain unknown.
Using gas chromatography-mass spectrometry (GC-MS), volatile compounds of kiwifruit were quantified in response to ethylene (ETH, 100 μl/l, 24 h, 20 °C) and 1-methylcyclopropene (1-MCP, 1 μl/l, 24 h, 20 °C). The results indicated that esters showed the most substantial changes enhanced by ethylene and were inhibited by 1-MCP. Correlations between RNA-seq results and concentrations of esters, constructed using Weighted Gene Co-Expression Network Analysis (WGCNA) indicated that three structural genes (fatty acid desaturase, AdFAD1; aldehyde dehydrogenase, AdALDH2; alcohol acyltransferase, AdAT17) had similar expression patterns that paralled the changes in total ester content, and AdFAD1 transcripts exhibited the highest correlation. In order to search for potential regulators for ester biosynthesis, 14 previously reported ethylene-responsive transcription factors (TFs) were included in the correlation analysis with esters and their biosynthetic genes. Using dual-luciferase assay, the in vivo regulatory activities of TFs on ester biosynthetic gene promoters were investigated and the results indicated that AdNAC5 and AdDof4 (DNA binding with one finger) trans-activated and trans-suppressed the AdFAD1 promoter.
The present study advanced the molecular basis of ripening-related ester biosynthesis in kiwifruit by identifying three biosynthetic related genes AdFAD1, AdALDH2 and AdAT17 by transcriptome analysis, and highlighted the function of two TFs by transactivation studies.
Prunus tomentosa
Thunb. has excellent nutritional, economic, and ornamental values with different fruit color. The red coloration of fruit is determined by anthocyanin pigmentation, which is an ...attractive trait for consumers. However, the mechanisms underlying fruit color formation in the
P. tomentosa
cherry are not well understood. In this research, the pigmentation patterns in red-color
P. tomentosa
(RP) fruit and white-color
P. tomentosa
(WP) were evaluated. Anthocyanin content in matured RP fruit was significantly abundant compared with WP fruit. Metabolomic profiling revealed that pelargonidin 3-
O
-glucoside, cyanidin 3-
O
-rutinoside, and pelargonidin 3-
O
-rutinoside were the predominant anthocyanin compounds in the RP fruit, while, WP fruit had less anthocyanin compositions and lower level. Then, integrative analyses of transcriptome and metabolome identified 285 significant differentially expressed genes (DEGs) closely related to anthocyanin differentially expressed metabolites (DEMs). Among them, nine genes were involved in anthocyanin biosynthesis, transport and degradation pathway, including four biosynthesis genes (
PtPAL1
,
PtDFR
,
PtANS, and PtUFGT
), two transport genes (
PtGST11
,
PtABC10
), and three degradation genes (
PtPOD1
,
PtPOD16, PtPOD73
). Transcriptome data and real-time PCR showed that the transcript levels of biosynthesis and transport genes were significantly higher in RP than in WP, especially
PtANS, PtUFGT,
and
PtGST11
, suggesting they may play key roles in red-colored fruit formation. Meanwhile, the degradation-related genes
PtPOD1/16/73
took on exactly opposite trend, suggesting their potential effects on anthocyanin degradation. These results provide novel insights into color patterns formation mechanisms of cherries fruit, and the candidate key genes identified in anthocyanin biosynthesis, transport and degradation may provide a valuable resource for cherry breeding research in future.
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