Expression level and phenotype of <i>PAP1</i> activation plants.

<p>(A) qRT-PCR analysis of expression levels of <i>PAP1</i> (P903) T1 transgenic plants under 250 μmol·m^-2·s^-1 light intensity. (B) Seedling phenotypes of <i>Pap1</i>^<i>dcas9</i> -<i>D</i> on culture media. (C) One-month-old plants showing purple color in growth chamber (top), adaxial side showing purple color (bottom). <i>Actin 2</i> was used as an endogenous control. Scale bar, 1cm.</p

RNA-guided transcriptional activation via CRISPR/dCas9 mimics overexpression phenotypes in Arabidopsis

<div><p>Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR associated protein 9 (Cas9) system allows effective gene modification through RNA-guided DNA targeting. The Cas9 has undergone a series of functional alterations from the original active endonuclease to partially or completely deactivated Cas9. The catalytically deactivated Cas9 (dCas9) offers a platform to regulate transcriptional expression with the addition of activator or repressor domains. We redesigned a CRISPR/Cas9 activation system by adding the p65 transactivating subunit of NF-kappa B and a heat-shock factor 1 (HSF) activation domain to dCas9 bound with the VP64 (tetramer of VP16) activation domain for application in plants. The redesigned CRISPR/Cas9 activation system was tested in Arabidopsis to increase endogenous transcriptional levels of <u><i>p</i></u><i>roduction of</i> <u><i>a</i></u><i>nthocyanin</i> <u><i>p</i></u><i>igment 1</i> (<i>PAP1</i>) and <u><i>A</i></u><i>rabidopsis thaliana</i> <u><i>v</i></u><i>acuolar H</i>^<i>+</i>-<u><i>p</i></u><i>yrophosphatase</i> (<i>AVP1</i>). The expression of <i>PAP1</i> was increased two- to three-fold and the activated plants exhibited purple leaves similar to that of <i>PAP1</i> overexpressors. The <i>AVP1</i> gene expression was increased two- to five-fold in transgenic plants. In comparison to the wild type, <i>AVP1</i> activated plants had increased leaf numbers, larger single-leaf areas and improved tolerance to drought stress. The <i>AVP1</i> activated plants showed similar phenotypes to <i>AVP1</i> overexpressors. Therefore, the redesigned CRISPR/Cas9 activation system containing modified p65-HSF provides a simple approach for producing activated plants by upregulating endogenous transcriptional levels.</p></div

Flowering time, rosette leaf number and single-leaf area of <i>AVP1</i> (P904) activation plants.

<p>Flowering time, rosette leaf number and single-leaf area of <i>AVP1</i> (P904) activation plants.</p

qRT-PCR analysis of transient expression levels of <i>PAP1</i> (A) and <i>AVP1</i> (B) constructs in Arabidopsis mesophyll protoplasts.

<p><i>PAP1</i> (P902 and P903 each carrying a single target; P905 carrying double targets), <i>AVP1</i> (P900 and P901 each carrying a single target; P904 carrying double targets), empty vector (P886) and GFP (35S-GFP). <i>Actin 2</i> was used as an endogenous control (value = 1).</p

Schematic diagram of the procedure for constructing a binary vector (P886) for the transcriptional activation system.

<p>Schematic diagram of the procedure for constructing a binary vector (P886) for the transcriptional activation system.</p

Phenotype of WT and <i>AVP</i> activation (<i>Avp1</i>^<i>dcas9</i> -<i>D</i>) T2 plants before or after drought stress.

<p>(A) Plants grown under regular conditions. (B) Watering was stopped for seven days. (C) Three days after re-watering the plants.</p

Construction of Whole Genome Radiation Hybrid Panels and Map of Chromosome 5A of Wheat Using Asymmetric Somatic Hybridization

<div><p>To explore the feasibility of constructing a whole genome radiation hybrid (WGRH) map in plant species with large genomes, asymmetric somatic hybridization between wheat (<em>Triticum aestivum</em> L.) and <em>Bupleurum scorzonerifolium</em> Willd. was performed. The protoplasts of wheat were irradiated with ultraviolet light (UV) and gamma-ray and rescued by protoplast fusion using <em>B. scorzonerifolium</em> as the recipient. Assessment of SSR markers showed that the radiation hybrids have the average marker retention frequency of 15.5%. Two RH panels (RHPWI and RHPWII) that contained 92 and 184 radiation hybrids, respectively, were developed and used for mapping of 68 SSR markers in chromosome 5A of wheat. A total of 1557 and 2034 breaks were detected in each panel. The RH map of chromosome 5A based on RHPWII was constructed. The distance of the comprehensive map was 2103 cR and the approximate resolution was estimated to be ∼501.6 kb/break. The RH panels evaluated in this study enabled us to order the ESTs in a single deletion bin or in the multiple bins cross the chromosome. These results demonstrated that RH mapping via protoplast fusion is feasible at the whole genome level for mapping purposes in wheat and the potential value of this mapping approach for the plant species with large genomes.</p> </div

MOESM5 of Switchgrass SBP-box transcription factors PvSPL1 and 2 function redundantly to initiate side tillers and affect biomass yield of energy crop

Additional file 5: Figure S3.  Expression levels of PvSPL1 in PvSPL1SRDX and rPvSPL1 overexpressing transgenic switchgrass plants were revealed by qRT-PCR. Switchgrass Ubq2 was used as the reference for normalization. PvSPL1: sum of exo- and endo-PvSPL1 transcript versions

Application of "Problem Solving" in Religious Educatiion

Anglická anotace (English annotation) Application of the "Problem solving" to the Religious education This dissertation work deals with the practical application of teaching methods "problem solving" in the lessons of religion with all its pros and cons. It deals with issues in the teaching of religious education 8th classes of elementary schools, using only the Czech language and suggested topics, which are set out in the curriculum of religious education Roman Catholic Church in 2004, for use in the diocese of Hradec Králové. The first part is devoted to learning theory, regulations, curriculum and teaching plans of the relevant legislation with regard to the specifics and the territorial limits. The following section presents the individual applicable methods in the "problem solving" with respect to the development of key competencies of students. And in the last section are selected appropriate methods discussed in details and presented on topics discussed in the teaching of religious education lessons. The course of instruction and the subsequent reactions of students and teachers alike are drawn appropriate recommendations to the methods already fully applicable. In conclusion, the proper methods for teaching religious education in the 8th elementary school classroom are described in detail for any..

Individual marker retention frequency and donor DNA retention frequency of wheat in radiation hybrids.

<p>A: Individual marker retention frequencies in 155 radiation hybrids derived from seven asymmetric hybridization combinations. Forty-two microsatellite loci across the wheat genome are listed to the left of the figure. B–D: Donor DNA retention frequency of wheat in hybrids. B: Average donor DNA retention frequencies of A, B, D genome of wheat in each hybridization combination; C: Average donor DNA retention frequencies of seven chromosome groups of wheat in each of hybridization combinations; D: Average donor DNA retention frequencies of seven asymmetric hybridization combinations and control combination.</p