Cloning, Structural Characterization, and Phylogenetic Analysis of Flower MADS-Box Genes from Crocus (Crocus sativusL.)

Crocus (Crocus sativusL.) is a crop species cultivated for its flowers and, more specifically, for its red stigmas. The flower of crocus is bisexual and sterile, since crocus is a triploid species. Its perianth consists of six petaloid tepals: three tepals in whorl 1 (outer tepals) and three tepals in whorl 2 (inner tepals). The androecium consists of three distinct stamens and the gynoecium consists of a single compound pistil with three carpels, a single three-branched style, and an inferior ovary. The dry form of the stigmas constitutes the commercial saffron used as a food additive, in the coloring industry, and in medicine. In order to uncover and understand the molecular mechanisms controlling flower development in cultivated crocus and its relative wild progenitor species, and characterize a number of crocus flower mutants, we have cloned and characterized different, full-length, cDNA sequences encoding MADS-box transcription factor proteins involved in flower formation.Here we review the different methods followed or developed for obtaining these sequences involving conventional 5' 3' RACE, as well as newly developed methods from our group, named Rolling Circle Amplification – RACE (RCA-RACE) and its modification named familyRCA-RACE (famRCA-RACE). Furthermore, the characteristics of the protein structure and their common and specific domains for each type of MADS-box transcription factors in this lower nongrass monocot belonging to the Iridaceae family are described. Finally, a phylogenetic tree of all the MADS-box sequences available in our lab is presented and discussed in relation to other data from studies of species of the Iridaceae group and closely related families from an evolutionary perspective. The structural and phylogenetic analyses are based on both published and unpublished data.

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Identification and Characterization of Two Novel Geminiviruses Associated with Paper Mulberry (Broussonetia papyrifera) Leaf Curl Disease

Plant Disease ◽

10.1094/pdis-12-19-2597-re ◽

2020 ◽

Vol 104 (11) ◽

pp. 3010-3018 ◽

Cited By ~ 1

Author(s):

Yuanjian Qiu ◽

Song Zhang ◽

Haodong Yu ◽

Zhiyou Xuan ◽

Liu Yang ◽

...

Keyword(s):

High Throughput Sequencing ◽

Close Association ◽

Phylogenetic Analyses ◽

Rolling Circle Amplification ◽

Field Investigation ◽

Broussonetia Papyrifera ◽

New Members ◽

Rolling Circle ◽

Mulberry Leaf ◽

Leaf Curl

Paper mulberry (Broussonetia papyrifera) is a perennial woody plant used as source material for Cai Lun paper making, in traditional Chinese medicine, and as livestock feed. To identify the presence of viruses in paper mulberry plants affected by a disease with leaf curl symptoms, high-throughput sequencing of total RNA was performed. Analysis of transcriptome libraries allowed the reconstruction of two geminivirus-like genomes. Rolling-circle amplification and PCR with back-to-back primers confirmed the presence of two geminiviruses with monopartite genomes in these plants, with the names paper mulberry leaf curl virus 1 and 2 (PMLCV-1 and PMLCV-2) proposed. The genomes of PMLCV-1 (3,056 nt) and PMLCV-2 (3,757 to 3,763 nt) encode six proteins, with the V4 protein of PMLCV-1 and the V3 proteins of both viruses having low similarities to any known protein in databases. Alternative splicing of an intron, akin to that of mastre-, becurto-, capula-, and grabloviruses, was identified by small RNA (sRNA)-seq and RNA-seq reads mapping to PMLCV-1 and PMLCV-2 antisense transcripts. Phylogenetic analyses and pairwise comparisons showed that PMLCV-1 and PMLCV-2 are most closely related to, but distinct from, two unassigned geminiviruses, citrus chlorotic dwarf associated virus and mulberry mosaic dwarf associated virus, suggesting that they are two new members of the family Geminiviridae. Field investigation confirmed the close association of the two viruses with leaf curl symptoms in paper mulberry plants and that coinfection can aggravate the symptoms.

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Rolling Circle Amplification (RCA)-Mediated Genome-Wide ihpRNAi Mutant Library Construction in Brassica napus

International Journal of Molecular Sciences ◽

10.3390/ijms21197243 ◽

2020 ◽

Vol 21 (19) ◽

pp. 7243

Author(s):

Shengbo Zhao ◽

Junling Luo ◽

Xinhua Zeng ◽

Keqi Li ◽

Rong Yuan ◽

...

Keyword(s):

Brassica Napus ◽

Functional Genomics ◽

Insertional Mutagenesis ◽

Molecular Mechanisms ◽

Target Genes ◽

Agronomic Traits ◽

Rolling Circle Amplification ◽

Functional Characterization ◽

Rolling Circle ◽

Genome Wide

With the successful completion of genomic sequencing for Brassica napus, identification of novel genes, determination of functions performed by genes, and exploring the molecular mechanisms underlying important agronomic traits were challenged. Mutagenesis-based functional genomics techniques including chemical, physical, and insertional mutagenesis have been used successfully in the functional characterization of genes. However, these techniques had their disadvantages and inherent limitations for allopolyploid Brassica napus, which contained a large number of homologous and redundant genes. Long intron-spliced hairpin RNA (ihpRNA) constructs which contained inverted repeats of the target gene separated by an intron, had been shown to be very effective in triggering RNAi in plants. In the present study, the genome-wide long ihpRNA library of B. napus was constructed with the rolling circle amplification (RCA)-mediated technology. Using the phytoene desaturase (PDS) gene as a target control, it was shown that the RCA-mediated long ihpRNA construct was significantly effective in triggering gene silence in B. napus. Subsequently, the resultant long ihpRNA library was transformed into B. napus to produce corresponding RNAi mutants. Among the obtained transgenic ihpRNA population of B. napus, five ihpRNA lines with observable mutant phenotypes were acquired including alterations in the floral model and the stamen development. The target genes could be quickly identified using specific primers. These results showed that the RCA-mediated ihpRNA construction method was effective for the genome-wide long ihpRNA library of B. napus, therefore providing a platform for study of functional genomics in allopolyploid B. napus.

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Discovery of a novel Torque teno sus virus species: genetic characterization, epidemiological assessment and disease association

Journal of General Virology ◽

10.1099/vir.0.045518-0 ◽

2012 ◽

Vol 93 (12) ◽

pp. 2682-2691 ◽

Cited By ~ 19

Author(s):

Vivian Cornelissen-Keijsers ◽

Alexandra Jiménez-Melsió ◽

Denny Sonnemans ◽

Martí Cortey ◽

Joaquim Segalés ◽

...

Keyword(s):

Genetic Characterization ◽

Phylogenetic Analyses ◽

Rolling Circle Amplification ◽

Disease Association ◽

Virus Species ◽

Viral Dna ◽

Rolling Circle ◽

Viral Loads ◽

Wasting Syndrome ◽

Torque Teno Sus Virus

The study describes a novel Torque teno sus virus (TTSuV) species, provisionally named Torque teno sus virus k2b (TTSuVk2b), originally found in commercial pig sera by applying the rolling-circle amplification technique. Full-length sequences of TTSuVk2b were obtained, annotated and used in the phylogenetic analyses, which revealed that TTSuVk2b is a novel Anellovirus species within the genus Kappatorquevirus of the family Anelloviridae. Quantitative PCR techniques were developed to determine total TTSuV DNA quantities as well as the prevalence and viral DNA quantities of TTSuV1, TTSuVk2a and TTSuVk2b. The mean total TTSuV load in seven commercial sera was determined at 6.3 log10 DNA copies ml−1 of serum, with TTSuVk2b loads being the lowest at 4.5 log10 DNA copies ml−1 of serum. Subsequently, prevalence and loads of TTSuVs were determined in pig sera from 17 countries. TTSuVk2b prevalence ranged from 0 to 100 % with viral loads from 3.3 to 4.6 log10 copies ml−1 of sera. TTSuVk2a, so far the only species in the genus Kappatorquevirus, has been linked to an economically important swine disease, namely post-weaning multisystemic wasting syndrome (PMWS). Considering the grouping of TTSuVk2b in the same genus as TTSuVk2a, TTSuVk2b prevalence and viral DNA load were determined in PMWS-affected animals and healthy counterparts. This revealed that TTSuVk2a and TTSuVk2b are not only genetically related, but also that their viral loads in serum are elevated in PMWS animals compared with those of healthy pen mates. In summary, the present work describes a novel TTSuV species including its genetic characterization, epidemiological assessment and potential disease association.

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Genomic characterization of ten novel cutaneous human papillomaviruses from keratotic lesions of immunosuppressed patients

Journal of General Virology ◽

10.1099/vir.0.030593-0 ◽

2011 ◽

Vol 92 (7) ◽

pp. 1585-1594 ◽

Cited By ~ 35

Author(s):

Anja Köhler ◽

Marc Gottschling ◽

Kizzie Manning ◽

Mandy D. Lehmann ◽

Eric Schulz ◽

...

Keyword(s):

Phylogenetic Analyses ◽

Rolling Circle Amplification ◽

Organ Transplant ◽

Human Papillomaviruses ◽

Rolling Circle ◽

Non Melanoma Skin Cancer ◽

Immunosuppressed Patients ◽

Genomic Characterization ◽

Hpv Types

Viral warts from immunosuppressed organ transplant recipients (OTR) persist over years and may progress into non-melanoma skin cancer. The types of human papillomaviruses (HPV) in such lesions are different from that seen in the general population. A subset of these lesions is not infected with the classical wart-associated HPV types. In order to gain a better understanding of the HPV types in those lesions, we isolated ten novel HPVs from persisting keratotic lesions of immunosuppressed OTRs by rolling circle amplification and subsequent long-template PCR. Additionally, we sequenced and characterized the whole genome of the ten novel HPV types. Phylogenetic analyses revealed that nine HPV types belonged to the genus Gammapapillomavirus (γ-PV) and one to the genus Betapapillomavirus. In a phylogenetic analysis using L1 fragments of human and non-human PV types, primate papillomaviruses and our novel HPV types nested within the genus γ-PV in a highly polyphyletic pattern. This study significantly broadens the knowledge concerning the diversity and evolution of the poorly known γ-PV types.

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famRCA-RACE: A ROLLING CIRCLE AMPLIFICATION RACE FOR ISOLATING A FAMILY OF HOMOLOGOUS cDNAs IN ONE REACTION AND ITS APPLICATION TO OBTAIN NAC GENES TRANSCRIPTION FACTORS FROM CROCUS (CROCUS SATIVUS) FLOWER

Preparative Biochemistry & Biotechnology ◽

10.1080/10826068.2010.488507 ◽

2010 ◽

Vol 40 (3) ◽

pp. 177-187 ◽

Cited By ~ 2

Author(s):

Apostolos Kalivas ◽

Konstantinos Pasentsis ◽

Anagnostis Argiriou ◽

Nikos Darzentas ◽

Athanasios S. Tsaftaris

Keyword(s):

Transcription Factors ◽

Rolling Circle Amplification ◽

Crocus Sativus ◽

Rolling Circle

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THE FAMILY OF MADS-BOX TRANSCRIPTION FACTORS CONTROLLING FLOWER FORMATION IN CROCUS SATIVUS L.

Acta Horticulturae ◽

10.17660/actahortic.2010.850.16 ◽

2010 ◽

pp. 107-112 ◽

Cited By ~ 1

Author(s):

A.S. Tsaftaris ◽

K. Pasentsis ◽

A. Kalivas ◽

A. Argiriou ◽

A.N. Polidoros

Keyword(s):

Transcription Factors ◽

Crocus Sativus ◽

Flower Formation ◽

Mads Box ◽

The Family ◽

Crocus Sativus L

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Ferromagnetic Resonance Biosensor for Homogeneous and Volumetric Detection of DNA

10.26434/chemrxiv.5662315.v1 ◽

2017 ◽

Author(s):

Bo Tian ◽

Peter Svedlindh ◽

Mattias Strömberg ◽

Erik Wetterskog

Keyword(s):

Magnetic Nanoparticles ◽

Ferromagnetic Resonance ◽

Limit Of Detection ◽

Point Of Care ◽

Rolling Circle Amplification ◽

Resonance Field ◽

Isothermal Amplification ◽

Detection Sensitivity ◽

Serum Samples ◽

Rolling Circle

In this work, we demonstrate for the first time, a ferromagnetic resonance (FMR) based homogeneous and volumetric biosensor for magnetic label detection. Two different isothermal amplification methods, i.e., rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) are adopted and combined with a standard electron paramagnetic resonance (EPR) spectrometer for FMR biosensing. For RCA-based FMR biosensor, binding of RCA products of a synthetic Vibrio cholerae target DNA sequence gives rise to the formation of aggregates of magnetic nanoparticles. Immobilization of nanoparticles within the aggregates leads to a decrease of the net anisotropy of the system and a concomitant increase of the resonance field. A limit of detection of 1 pM is obtained with an average coefficient of variation of 0.16%, which is superior to the performance of other reported RCA-based magnetic biosensors. For LAMP-based sensing, a synthetic Zika virus target oligonucleotide is amplified and detected in 20% serum samples. Immobilization of magnetic nanoparticles is induced by their co-precipitation with Mg2P2O7 (a by-product of LAMP) and provides a detection sensitivity of 100 aM. The fast measurement, high sensitivity and miniaturization potential of the proposed FMR biosensing technology makes it a promising candidate for designing future point-of-care devices.

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