Relative transcript levels of putative genes after paraquat treatment.

A. Gene transcript levels in susceptible (S) and resistant (R) goosegrass at the indicated times after paraquat treatment. Transcripts were detected by real-time PCR using total RNA extracted from 10-d-old seedlings sprayed with 10/HM paraquat. Error bars represent the SD (n = 3). B. Amplification products of 4 putative genes from goosegrass by RT-PCR. For the M:DL 2000 marker, “-” indicates the negative control. R0, R30, R60, R90, R120, and R180 indicate samples of R goosegrass seedlings taken 0, 30, 60, 90, 120 and 180 min after paraquat treatment, while S0, S30, S60, S90, S120 and S180 indicate samples of S goosegrass seedlings taken 0, 30, 60, 90, 120 and 180 min after paraquat treatment, respectively.</p

Polyamine levels in susceptible (S) and resistant (R) goosegrass after a paraquat treatment.

Putrescine (A), spermidine (B), and spermine (C) were extracted from shoots of R goosegrass (black bar) and S goosegrass (white bar) seedlings 0, 30, 60, 90, 120, and 180 min after spraying with paraquat and then quantified by HPLC. Different lowercase letters indicate significant differences at P<0.05 (t-test).</p

Primers used to amplify four putative genes by real-time PCR.

Primers used to amplify four putative genes by real-time PCR.</p

Biological analysis of four putative genes^*.

Biological analysis of four putative genes*.</p

One-Step Synthesis of Uniform Double-Shelled Polystyrene/Poly(<i>o</i>‑toluidine) Composite Hollow Spheres

Uniform double-shelled polystyrene/poly­(<i>o</i>-toluidine) (PS/POT) composite hollow spheres with tunable shell thickness of the POT layer have been successfully synthesized by a simple method. POT was directly coated onto the surface of negatively charged PS template spheres, which were synthesized by soap-free emulsion polymerization. Surprisingly, the resultant spheres show a double-shelled hollow structure with PS as an inner wall and POT as an exterior shell. In comparison to conventional methods, the benefits of this route are that neither organic solvents nor high-temperature calcinations were used to remove the PS template. The surface morphology, the shell thickness, and the compositions of the double-shelled spheres were systematically characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) spectroscopy. On the basis of our interpretation of experimental results, a mechanism for the formation of the double-shelled PS/POT composite hollow spheres is proposed

Cloning and Analysis of a Large Plasmid pBMB165 from <i>Bacillus thuringiensis</i> Revealed a Novel Plasmid Organization

<div><p>In this study, we report a rapid cloning strategy for large native plasmids via a contig linkage map by BAC libraries. Using this method, we cloned a large plasmid pBMB165 from <i>Bacillus thuringiensis</i> serovar <i>tenebrionis</i> strain YBT-1765. Complete sequencing showed that pBMB165 is 77,627 bp long with a GC-content of 35.36%, and contains 103 open reading frames (ORFs). Sequence analysis and comparison reveals that pBMB165 represents a novel plasmid organization: it mainly consists of a pXO2-like replicon and mobile genetic elements (an inducible prophage BMBTP3 and a set of transposable elements). This is the first description of this plasmid organization pattern, which may result from recombination events among the plasmid replicon, prophage and transposable elements. This plasmid organization reveals that the prophage BMBTP3 may use the plasmid replicon to maintain its genetic stability. Our results provide a new approach to understanding co-evolution between bacterial plasmids and bacteriophage.</p> </div

Identifying BMBTP3 among the total induced phage DNA from B.

<p><b><i>thuringiensis</i> strain YBT-1765</b>. <b>A</b>. Southern hybridization with a replication-associated protein gene specific probe (probe-rep). Lane 1, the total plasmid DNA extracted from YBT-1765; Lane 2, digested total plasmid DNA by <i>Hin</i>dIII; Lane 3, digested total induced phage DNA by <i>Hin</i>dIII; Lane 4, digested total plasmid DNA by <i>Hin</i>cII; Lane 5, digested total induced phage DNA by <i>Hin</i>cII; Lane 6, digested total plasmid DNA by <i>Hpa</i>I; Lane 7, digested total induced phage DNA by <i>Hpa</i>I. <b>B</b>. Southern hybridization with a phage terminase large subunit gene specific probe (probe-term). Lane 1, the total plasmid DNA extracted from YBT-1765; Lane 2, digested total plasmid DNA by <i>Hin</i>dIII; Lane 3, digested total induced phage DNA by <i>Hin</i>dIII; Lane 4, digested total plasmid DNA by <i>Eco</i>RV; Lane 5, digested total induced phage DNA by <i>Eco</i>RV. The sizes of the signal bands are labeled with arrows. In each lane for total plasmids and digested products we loaded 0.7 μg plasmid DNA (lanes 1, 2, 4, 6 in Figures 3A and 1, 2, 4 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081746#pone-0081746-g003" target="_blank">Figure 3B</a>), and for the purified phage DNA and digested products, we loaded 1.3 μg in each lane (lanes 3, 5, 7 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081746#pone-0081746-g003" target="_blank">Fig. 3A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081746#pone-0081746-g003" target="_blank">3</a>, 5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081746#pone-0081746-g003" target="_blank">Fig. 3B</a>). <b>C</b>. The schematic drawing shows the structure of the restriction fragments with the ORF015 (probe-rep), ORF120 (probe-term) and the predicted cos site. The dashed line denotes the DNA of pBMB165, and the sizes of fragment digested by the restriction enzymes and the predicted cos site.</p

Comparison of pBMB165 and homologous plasmids and phages by Easyfig alignment.

<p>Coding Sequences (CDSs) are represented by colored arrows. Predicted functions/homologies are indicated by the color key featured below. The pXO2-like replicon is highlighted with a green frame. Color coding for the genes is as follows: olive green, plasmid replication; deep green, prophage replication; deep yellow, plasmid stabilization system; orange, regulatory; red, a predicted camelysin; blue, mobile DNA; purple, phage related; grey, hypothetical protein; midnight blue, conjugation-related proteins; wine, capsule synthesis related proteins; and brown, other determinants. Highly conserved segments of the plasmids and phages are paired by shaded regions, with the darker shading reflecting a greater amino acid identity, from 66% (A) or 63% (B) to 100%. The regions outside the shaded regions lack homology between plasmids and phages. The outer scale is marked in kilobases.</p

Circular representation of plasmid pBMB165 and graphical representation of the annotation and the structure of the prophage BMBTP3.

<p>The inner circle represents the GC bias [(G - C)/(G + C)], with positive and negative values in reddish brown and cobalt blue, respectively; the second circle represents the GC-content, with positive and negative values in grey and black, respectively; and the outer circle represents the predicted genes on the reverse and forward arrows. The pXO2-like replicon is highlighted with a green arching frame. Regions of transposon and prophage BMBTP3 are annotated beside the corresponding arrows and separated by straight lines. The main functional genes of BMBTP3 are annotated above the extended corresponding arrows at the bottom. Different structural and functional regions are annotated and separated by vertical lines. Color coding for the genes is as follows: olive green, plasmid replication; deep green, prophage replication; deep yellow, plasmid stabilization system; orange, regulatory; red, a predicted camelysin; blue, mobile DNA; purple, phage related; grey, hypothetical protein. The outer scale is marked in kilobases. </p