Multiple sequence alignment and phylogenetic analysis of <i>BnPRP1</i>.

<p>(A) Unrooted Phylogenetic tree of BnPRP1 and its homologous proteins. Dendrogram obtained using neighbor-joining analysis based on the proportion (p-distance) of aligned amino acid sites of the full-length peptide sequences. BnPRP1 is marked with a solid triangle. Numbers at the base of each clade correspond to the bootstrap means of 1000 replications. Organism’s common or taxonomic names are shown in parenthesis. The Genbank accession numbers/Database accession numbers for the proteins/peptides are as follows: <i>Arabidopsis thaliana</i> (NP_197850.2); <i>Capsella rubella</i> (XP_006289819); <i>Camelina sativa</i> (XP_010421292.1); <i>Arabidopsis lyrata</i> (XP_00287212); <i>Eutrema salsugineum</i> (XP_0063947); <i>Brassica napus</i> (<i>Brassica napus</i> Database accession No. GSBRNA2T00120298001); <i>Brassica oleracea</i> (bolbase accession No: Bol016440); <i>Brassica rapa</i> (brad accession No: XP_009130006.1); <i>Brassica napus</i> (<i>Brassica napus</i> Database accession No:GSBRNA2T00147553001); <i>Brassica rapa</i> (brad accession No: XP_009150986.1); <i>Brassica napus</i> (GSBRNA2T00133542001); <i>Brassica oleracea</i> (bolbase accession No: Bol022411); <i>Brassica napus</i> (<i>Brassica napus</i> Database accession No: GSBRNA2T00120360001); SP-B (APD accession No: AP00889); BacFL31(APD accession No: AP02346); bumblebee_Abaecins (APD accession No: AP01215); honeybee_Abaecins (APD accession No: AP00002). (B) Multiple alignments of BnPRP1 and its homologs. Multiple alignments of BnPRP1 and its homologs identical amino acid residues were black-shaded, PPT motif of BnPRP1 is shown in box.</p

Minimal concentrations of <i>BnPRP1</i> required for complete growth inhibition.

<p>^aResults are the mean values obtained from three independent measurements.</p><p>Minimal concentrations of <i>BnPRP1</i> required for complete growth inhibition.</p

Response of <i>BnPRP1</i> Expression to <i>S</i>. <i>sclerotiorum</i> inoculation.

<p>Quantitative real-time PCR was used to measure <i>BnPRP1</i> gene expression in the leaves and stems of six <i>Brassica napus</i> cultivars: four disease-resistant (Zhongshuang 9, M083, Zhongshuang 11, and Zhongyou 821) and two susceptible cultivars (84039 and 888–5). All values are the means obtained from three biological replicates and their respective standard deviations.</p

Structural data of <i>BnPRP1</i> obtained from CD.

<p>^aPBS, pH 7.4</p><p>TFE⁺, trifluoroethanol; MeOH, methanol; EtOH, ethanol</p><p>Structural data of <i>BnPRP1</i> obtained from CD.</p

CD spectra of <i>BnPRP1</i> in different solvents (A) and in the presence of 0–75% (v/v) TFE (B).

<p>CD spectra of <i>BnPRP1</i> in different solvents (A) and in the presence of 0–75% (v/v) TFE (B).</p

Agarose gel electrophoresis detection of the overlap PCR product from the <i>BnPRP1</i> gene and the recombinant plasmids pET30a/His-EDDIE-GFP and pET30a-EDDIE-BnPRP1.

<p>(A) Lane M, 100 bp DNA marker; Lanes 1–2, Overlap PCR products from the <i>BnPRP1</i> gene. (B) Lane M, 1 kb DNA marker; Lane 1, recombinant vector pET30a/His-EDDIE-BnPRP1; Lane 2, vector pET30a/His-EDDIE-GFP. <b>C:</b> Lane M, DL2000 DNA marker; Lane 1, positive control; Lane 2, negative control; Lanes 3 and 4, PCR detection of the <i>BnPRP1</i> gene.</p

DataSheet_1_Comprehensive evolutionary analysis of growth-regulating factor gene family revealing the potential molecular basis under multiple hormonal stress in Gramineae crops.docx

Growth-regulating factors (GRFs) are plant-specific transcription factors that contain two highly conserved QLQ and WRC domains, which control a range of biological functions, including leaf growth, floral organ development, and phytohormone signaling. However, knowledge of the evolutionary patterns and driving forces of GRFs in Gramineae crops is limited and poorly characterized. In this study, a total of 96 GRFs were identified from eight crops of Brachypodium distachyon, Hordeum vulgare, Oryza sativa L. ssp. indica, Oryza rufipogon, Oryza sativa L. ssp. japonica, Setaria italic, Sorghum bicolor and Zea mays. Based on their protein sequences, the GRFs were classified into three groups. Evolutionary analysis indicated that the whole-genome or segmental duplication plays an essential role in the GRFs expansion, and the GRFs were negatively selected during the evolution of Gramineae crops. The GRFs protein function as transcriptional activators with distinctive structural motifs in different groups. In addition, the expression of GRFs was induced under multiple hormonal stress, including IAA, BR, GA3, 6BA, ABA, and MeJ treatments. Specifically, OjGRF11 was significantly induced by IAA at 6 h after phytohormone treatment. Transgenic experiments showed that roots overexpressing OjGRF11 were more sensitive to IAA and affect root elongation. This study will broaden our insights into the origin and evolution of the GRF family in Gramineae crops and will facilitate further research on GRF function.</p

SDS-PAGE analysis of recombinant His-EDDIE-BnPRP1 expressed in <i>E</i>. <i>coli</i> BL 21 (DE3) and Tricine-SDS-PAGE analysis of BnPRP1 renaturation.

<p>(A) SDS-PAGE analysis of recombinant His-EDDIE-BnPRP1. Lane M, protein molecular weight maker; Lanes 2, precipitation of His-EDDIE-BnPRP1; Lane 1, <i>E</i>. <i>coli</i> BL 21 (DE3) cells transformed with pET30a plasmid as a control. Lane 3, purified His-EDDIE-BnPRP1. (B) Tricine-SDS-PAGE analysis of BnPRP1 renaturation. Lane M, protein molecular weight marker; Lanes 1, 2, refolded BnPRP1 after 8 h.</p