Gene silencing induced by RNA1 16K gene mutants of Tobacco rattle virus on Nicotiana benthamiana

Virusinduced gene silencing (VIGS) vectors based on tobacco rattle virus (TRV) are now widely used for characterizing the function of plant genes. However, previous TRV vectors using RNA2 to carry the targeted gene sequence had difficulties to induce gene silencing on some plant species (Gerbera hybrida etc.) due to the obstacle of RNA2 movement. To achieve efficient gene silencing in those species, it is necessary to develop new TRV vectors, in which the targeted gene will be included in TRV RNA1 and the 16K gene will be replaced. Based on TRV RNA1, two new VIGS vectors M1 and M2 were developed through deletion part of 16K gene. Another mutant 16Kstop was also constructed to carry an early terminator in the 4th codon of 16K gene. The infectivity and gene silencing efficiency of the new constructs were assessed through a series of infection experiments. It was found that the infectivity of M1 and M2 was lower than wild TRV RNA1. M1 and M2 could induce PDS gene silencing on Nicotiana benthamiana, but their gene silencing efficiency was limited as compared with previous TRV VIGS vectors in which the PDS gene fragment was contained in RNA2. We also found that the 16K gene sequence, rather than the 16K protein, was required for efficient virus movement and accumulation

Developement and Application of Tobacco Rattle Virus Induced Gene Silencing in Gerbera hybrida

RNA silencing is a conserved mechanism that occurs in a broad range of eukaryotes, which is regulated by small RNAs (sRNAs). RNA silencing operates to control gene expression and maintain genome integrity. Virus-induced gene silencing (VIGS) in plants is a natural antivirus mechanism that has adapted from the general RNA silencing system. To counter the antivirus RNA silencing, plant viruses have evolved to encode viral suppressors of RNA silencing (VSRs). Nowadays VIGS is usually referred to as the technology that uses recombinant viruses to knock down the expression of plant endogenous genes. Gerbera hybrida (gerbera) is a model species in the family of Asteraceae. As a highly heterozygous species, gerbera lacks efficient functional genetic approaches other than gene transfer. The aim of the present study was to develop a Tobacco rattle virus (TRV, genus Tobravirus) induced gene silencing system for gerbera, and use TRV VIGS to characterize functions of chalcone synthase (CHS) encoding genes in the plant. Preliminary VIGS experiments on the cultivar Terraregina, by syringe infiltration and applying previously developed TRV vectors, did not result in visible VIGS phenotypes due to the absent of TRV RNA2 in the up non-infiltrated leaves. Consequently, I first aimed to study the mechanism of TRV VIGS, and tried to develop new VIGS vectors based on TRV RNA1. I investigated the role of two important TRV proteins of the 16K VSR and the 29K movement protein (MP) on TRV infection and TRV VIGS, and developed TRV RNA1 based VIGS vectors. For accomplishing this, a series of TRV RNA1 mutants have been constructed to disrupt the 16K, or to replace its 29K with Tobacco mosaic virus (TMV, genus Tobamovirus) 30K MP. TRV RNA1 vector, carrying a fragment of the gene encoding Nicotiana benthamiana PDS to replace part of the 16K sequence, induced PDS gene silencing systemically in N. benthamiana. However, this has found to be less efficiently than the original TRV VIGS system when the wild-type RNA1 and RNA2:PDS were used. The infection experiments demonstrated that 16K was required for TRV long distance movement, and helped in maintaining the integrity of the TRV RNA2 genome. In addition, TRV 29K alone did not suppress RNA silencing in the co-infiltration assay, but it could suppress RNA silencing in the context of RNA1 replication. TRV 29K may be the first VSR whose silencing suppression functions are found to be directly linked to viral replication. The original TRV vector system was finally adopted for VIGS in gerbera. TRV VIGS was optimized for gerbera by screening for TRV sensitive cultivars and by improving its inoculation methods. Intensive gene silencing phenotypes were achieved both in green tissues and in floral tissues, demonstrated by knocking down genes involved in isoprenoid biosynthesis (phytoene desaturase: GPDS; H and I subunits of Mg-chelatase: GChl-H and GChl-I), flower pigmentation (chalcone synthase: GCHS1), and flower development (GLOBOSA-like MADS domain transcription factor: GGLO1). Unexpectedly, a gerbera polyketide synthase encoding gene, G2PS1, that has no apparent connections to the carotenoid or chlorophyll biosynthesis, was knocked down by the photo-bleaching that was induced by the silencing of GPDS, GChl-H and GChl-I, or by the herbicide norflurazon. We have demonstrated for the first time that the using of VIGS in an Asteraceaeous species. Our data also suggested that the selection and use of a marker gene for VIGS should be strictly evaluated. A new CHS encoding gene, GCHS4, was characterized in gerbera. Together with the two previously identified GCHS1 and GCHS3, gerbera CHSs are represented by a three-gene family. Each gerbera CHS shows a distinct expression pattern. GCHS3 is particularly expressed in gerbera pappus. In partnership with the concomitantly expressed GCHS1, they are involved in the biosynthesis of colorless flavonoids. GCHS4 is the only CHS that is naturally expressed in the leaf petiole and inflorescence scape, and it is responsible for cyanidin biosynthesis in those tissues. GCHS4 is also the only CHS that was induced by environmental stresses in the leaf blade. Both GCHS1 and GCHS4 are markedly expressed in gerbera petals, and GCHS4 mRNA actually takes the majority of CHS mRNAs in the later stages of petal development. Nonetheless, VIGS experiments, by target silencing GCHS1 or GCHS4 independently, demonstrated that GCHS1 is the predominant functional CHS in gerbera petals. Thus, GCHS4 in gerbera petals seems to be regulated post-transcriptionally. In conclusion, the results of this study shed new light on the mechanism of TRV VIGS. The established TRV VIGS system provides a valuable tool for functional genomics in gerbera

Functional characterization and expression of GASCL1 and GASCL2, two anther-specific chalcone synthase like enzymes from Gerbera hybrida

The chalcone synthase superfamily consists of type III polyketidesynthases (PKSs), enzymes responsible for producing plant secondary metabolites with various biological and pharmacological activities. Anther-specific chalcone synthase-like enzymes (ASCLs) represent an ancient group of type III PKSs involved in the biosynthesis of sporopollenin, the main component of the exine layer of moss spores and mature pollen grains of seed plants. In the latter, ASCL proteins are localized in the tapetal cells of the anther where they participate in sporopollenin biosynthesis and exine formation within the locule. It is thought that the enzymes responsible for sporopollenin biosynthesis are highly conserved, and thus far, each angiosperm species with a genome sequenced has possessed two ASCL genes, which in Arabidopsis thaliana are PKSA and PKSB. The Gerbera hybrida (gerbera) PKS protein family consists of three chalcone synthases (GCHS1, GCHS3 and GCHS4) and three 2-pyrone synthases (G2PS1, G2PS2 and G2PS3). In previous studies we have demonstrated the functions of chalcone synthases in flavonoid biosynthesis, and the involvement of 2-pyrone synthases in the biosynthesis of antimicrobial compounds found in gerbera. In this study we expanded the gerbera PKS-family by functionally characterizing two gerbera ASCL proteins. In vitro enzymatic studies using purified recombinant proteins showed that both GASCL1 and GASCL2 were able to use medium and long-chain acyl-CoA starters and perform two to three condensation reactions of malonyl-CoA to produce tri- and tetraketide 2-pyrones, usually referred to as alpha-pyrones in sporopollenin literature. Both GASCL1 and GASCL2 genes were expressed only floral organs, with most expression observed in anthers. In the anthers, transcripts of both genes showed strict tapetum-specific localization. (C) 2016 Elsevier Ltd. All rights reserved.Peer reviewe

Virus-induced gene silencing for Asteraceae-a reverse genetics approach for functional genomics in Gerbera hybrida

Peer reviewe

Comprehensive Analysis and Functional Studies of WRKY Transcription Factors in Nelumbo nucifera

The WRKY family is one of the largest transcription factor (TF) families in plants and plays central roles in modulating plant stress responses and developmental processes, as well as secondary metabolic regulations. Lotus (Nelumbo nucifera) is an aquatic crop that has significant food, ornamental and pharmacological values. Here, we performed an overview analysis of WRKY TF family members in lotus, and studied their functions in environmental adaptation and regulation of lotus benzylisoquinoline alkaloid (BIA) biosynthesis. A total of 65 WRKY genes were identified in the lotus genome and they were well clustered in a similar pattern with their Arabidopsis homologs in seven groups (designated I, IIa-IIe, and III), although no lotus WRKY was clustered in the group IIIa. Most lotus WRKYs were functionally paired, which was attributed to the recently occurred whole genome duplication in lotus. In addition, lotus WRKYs were regulated dramatically by salicilic acid (SA), jasmonic acid (JA), and submergence treatments, and two lotus WRKYs, NnWRKY40a and NnWRKY40b, were significantly induced by JA and promoted lotus BIA biosynthesis through activating BIA biosynthetic genes. The investigation of WRKY TFs for this basal eudicot reveals new insights into the evolution of the WRKY family, and provides fundamental information for their functional studies and lotus breeding

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

N/S co-doped carbon dots-based solution-gated graphene transistors (SGGT) for ultra-sensitive and selective determination of Hg (II) ions in real water and serum samples

The conventional methods for detecting Hg2+ ions are generally impaired by low sensitivity, a high limit of detection (LOD), and the long detection time required. In this work, we develop a novel solution gated graphene transistor (SGGT) based on carbon dots doped with N and S elements (N/S CDs) as gate electrode probes. The prepared N/S CDs, which are 3–5 nm in diameter, are first modified at the gate of the SGGT through covalent bonding that specifically recognizes Hg2+ ions. The SGGT is then used as a Hg2+ sensor, exhibiting good selectivity, ultrahigh sensitivity, good reproducibility, and a short detection time. The SGGT sensor has an excellent linear detection range, from 0.1 fM to 1 nM. The LOD can reach 28 aM, which is far lower than that of conventional detection methods. Moreover, the SGGT sensor also has a rapid response time of ∼ 40 s. Finally, the ability to analyze real water samples and serum samples is demonstrated

Achieving Solar‐Thermal‐Electro Integration Evaporator Nine‐Grid Array with Asymmetric Strategy for Simultaneous Harvesting Clean Water and Electricity

Abstract Water evaporation is a ubiquitous and spontaneous phase transition process. The utilization of solar‐driven interface water evaporation that simultaneously obtains clean water and power generation can effectively alleviate people's concerns about fresh water and energy shortages. However, it remains a great challenge to efficiently integrate the required functions into the same device to reduce the complexity of the system and alleviate its dependence on solar energy to achieve full‐time operation. In this work, a multifunctional device based on reduced graphene oxide (RGO)/Mn3O4/Al2O3 composite nanomaterials is realized by an asymmetric strategy for effective solar‐thermal‐electro integration that can induce power generation by water evaporation in the presence/absence of light. Under one sun irradiation, the solar‐driven evaporation rate and output voltage are 1.74 kg m−2 h−1 and 0.778 V, respectively. More strikingly, the nine‐grid evaporation/power generation array integrated with multiple devices in series has the advantages of small volume, large evaporation area, and high power generation, and can light up light‐emitting diodes (LEDs), providing the possibility for large‐scale production and application. Based on the high photothermal conversion efficiency and power production capacity of the RGO/Mn3O4/Al2O3 composite evaporation/generator, it will be a promising energy conversion device for future sustainable energy development and applications

Unamplified and Real‐Time Label‐Free miRNA‐21 Detection Using Solution‐Gated Graphene Transistors in Prostate Cancer Diagnosis

Abstract The incidence of prostate cancer (PCa) in men globally increases as the standard of living improves. Blood serum biomarker prostate‐specific antigen (PSA) detection is the gold standard assay that do not meet the requirements of early detection. Herein, a solution‐gated graphene transistor (SGGT) biosensor for the ultrasensitive and rapid quantification detection of the early prostate cancer‐relevant biomarker, miRNA‐21 is reported. The designed single‐stranded DNA (ssDNA) probes immobilized on the Au gate can hybridize effectively with the miRNA‐21 molecules targets and induce the Dirac voltage shifts of SGGT transfer curves. The limit of detection (LOD) of the sensor can reach 10−20 M without amplification and any chemical or biological labeling. The detection linear range is from 10−20 to 10−12 M. The sensor can realize real‐time detection of the miRNA‐21 molecules in less than 5 min and can well distinguish one‐mismatched miRNA‐21 molecule. The blood serum samples from the patients without RNA extraction and amplification are measured. The results demonstrated that the biosensor can well distinguish the cancer patients from the control group and has higher sensitivity (100%) than PSA detection (58.3%). Contrastingly, it can be found that the PSA level is not directly related to PCa