Journal: PLoS ONE

Article Title: Enhanced Gene Expression Rather than Natural Polymorphism in Coding Sequence of the OsbZIP23 Determines Drought Tolerance and Yield Improvement in Rice Genotypes

doi: 10.1371/journal.pone.0150763

Figure Lengend Snippet: (A) PCR amplification of OsbZIP23 CDS from selected genotypes using gene-specific primers. Lanes 1–15 represents O . rufipogon , O . nivara , Vandana, Swarna, Nagina22, Manipuri, HRC300, IR20, IR36, IR64, IR72, sorghum, maize, Brachypodium and bajra respectively. Lane M- Hin fI digested pUC18 plasmid as a standard molecular weight marker. (B) Allelic polymorphism in the CDS of OsbZIP23 gene across selected rice genotypes is represented by multiple sequence alignment in Jalview software.

Article Snippet: Affinity purified polyclonal antibody raised against OsbZIP23 protein in rabbit (catalog no- E580041-A-SE, EnoGene Biotech, USA) was used as primary antibody (1: 1,000 dilution) and monoclonal plant anti-actin antibody (Sigma, catalog no.-A0480) was used as loading control (1:500 dilution).

Techniques: Amplification, Plasmid Preparation, Molecular Weight, Marker, Sequencing, Software

Journal: PLoS ONE

Article Title: Enhanced Gene Expression Rather than Natural Polymorphism in Coding Sequence of the OsbZIP23 Determines Drought Tolerance and Yield Improvement in Rice Genotypes

doi: 10.1371/journal.pone.0150763

Figure Lengend Snippet: (A) Relative expression level of OsbZIP23 gene in different tissues of an upland indica rice genotype, Vandana. Real time PCR analysis of OsbZIP23 transcript in vegetative (B) and reproductive (grain filling) stage (C) , in 11 selected rice genotypes grown under before stress (BS), after stress (AS) and after recovery (AR) condition as described in method section. Rice polyubiquitin1 ( OsUbi1 ) gene was taken as internal reference. Data bars represent the mean ±SD of triplicate measurement. Statistical analysis of Student’s t test indicated significant differences (* P<0.05, **P<0.01). (D) Southern blot showing the single copy of endogenous OsbZIP23 gene in 11 selected rice genotypes. Lanes 1–11 represent O . rufipogon , O . nivara , Vandana, IR20, Swarna, IR36, IR64, IR72, Nagina22, Manipuri, HRC300 rice genotypes, respectively.

Article Snippet: Affinity purified polyclonal antibody raised against OsbZIP23 protein in rabbit (catalog no- E580041-A-SE, EnoGene Biotech, USA) was used as primary antibody (1: 1,000 dilution) and monoclonal plant anti-actin antibody (Sigma, catalog no.-A0480) was used as loading control (1:500 dilution).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Southern Blot

Journal: PLoS ONE

Article Title: Enhanced Gene Expression Rather than Natural Polymorphism in Coding Sequence of the OsbZIP23 Determines Drought Tolerance and Yield Improvement in Rice Genotypes

doi: 10.1371/journal.pone.0150763

Figure Lengend Snippet: (A) PCR amplification of the OsbZIP23 gene promoter region from two selected rice genotypes using gene-specific primers. Lanes 1–2 represent O . rufipogon and IR20, respectively. Lane M- Hin fI digested pUC18 plasmid as a standard molecular weight marker. (B) Alignment of nucleotide sequences and distribution of major cis -regulatory elements in the OsbZIP23 promoter of O . rufipogon and IR20.

Article Snippet: Affinity purified polyclonal antibody raised against OsbZIP23 protein in rabbit (catalog no- E580041-A-SE, EnoGene Biotech, USA) was used as primary antibody (1: 1,000 dilution) and monoclonal plant anti-actin antibody (Sigma, catalog no.-A0480) was used as loading control (1:500 dilution).

Techniques: Amplification, Plasmid Preparation, Molecular Weight, Marker

Journal: PLoS ONE

Article Title: Enhanced Gene Expression Rather than Natural Polymorphism in Coding Sequence of the OsbZIP23 Determines Drought Tolerance and Yield Improvement in Rice Genotypes

doi: 10.1371/journal.pone.0150763

Figure Lengend Snippet: (A) Southern blot analysis of T 0 transformants of OEN and OER developed with the genetic construct of OsbZIP23 CDS from O . rufipogon and O . nivara . The genomic DNA was digested with Hind III and probed with the 739 bp OsbZIP23 CDS. (B) Southern blot analysis of T 0 transformants of RNAi developed with gene silencing construct using Hind III digested genomic DNA and probed with 739 bp OsbZIP23 CDS. Lane NT- non-transgenic, Lane M- molecular weight marker. (C) Real time PCR analysis showing relative expression level of OsbZIP23 in three OE lines, two RNAi lines and non-transgenic (NT) plants in vegetative stage, where rice polyubiquitin1 ( OsUbi1 ) gene was taken as internal reference. Data bars represent the mean ±SD of triplicate measurement. Statistical analysis of Student’s t test indicated significant differences (* P<0.05, **P<0.01). (D) Western blot analysis showing the expression level of OsbZIP23 protein in three OE lines, two RNAi lines and NT plant (upper panel). Equal loading of protein was confirmed by using β -actin antibody (lower panel).

Article Snippet: Affinity purified polyclonal antibody raised against OsbZIP23 protein in rabbit (catalog no- E580041-A-SE, EnoGene Biotech, USA) was used as primary antibody (1: 1,000 dilution) and monoclonal plant anti-actin antibody (Sigma, catalog no.-A0480) was used as loading control (1:500 dilution).

Techniques: Southern Blot, Construct, Transgenic Assay, Molecular Weight, Marker, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Enhanced Gene Expression Rather than Natural Polymorphism in Coding Sequence of the OsbZIP23 Determines Drought Tolerance and Yield Improvement in Rice Genotypes

doi: 10.1371/journal.pone.0150763

Figure Lengend Snippet: (A) Water loss rates and (B) relative water contents of detached leaves, at the five-leaf stage amongst OsbZIP23 OE lines, RNAi lines and NT plants. Measurement of (C) proline and (D) soluble sugar contents in OsbZIP23 OE lines, RNAi lines and NT plants, after extraction from leaf tissues before and after water stress. All the results were based on three independent experiments. Data bars represent the mean ±SD of triplicate measurement. A statistical analysis by Student’s t-test indicated significant differences (*P<0.05, **P<0.01). (E) Measurement of MDA content in OsbZIP23 OE, RNAi lines and NT plants, after extraction from leaf tissue of rice plants before and after water stress. Data bars represent the mean ±SD of triplicate measurement. A statistical analysis by Student’s t-test indicated significant differences (*P<0.05, **P<0.01). (F) Detection of ROS by monitoring H 2 O 2 production in leaves of OsbZIP23 OE, RNAi lines and NT plants were visualized by staining with 3, 3 ׳ –diaminobenzidine (DAB) under well-watered (normal) and drought stress condition. (G) Production of O 2 − ions in leaves of OsbZIP23 OE, RNAi lines and NT plants were visualized by staining with nitro blue tetrazolium (NBT) under normal and drought stress condition. The results were based on three independent experiments; one set of result is represented here.

Article Snippet: Affinity purified polyclonal antibody raised against OsbZIP23 protein in rabbit (catalog no- E580041-A-SE, EnoGene Biotech, USA) was used as primary antibody (1: 1,000 dilution) and monoclonal plant anti-actin antibody (Sigma, catalog no.-A0480) was used as loading control (1:500 dilution).

Techniques: Extraction, Staining

Journal: PLoS ONE

Article Title: Enhanced Gene Expression Rather than Natural Polymorphism in Coding Sequence of the OsbZIP23 Determines Drought Tolerance and Yield Improvement in Rice Genotypes

doi: 10.1371/journal.pone.0150763

Figure Lengend Snippet: (A) OsRab16B , (B) OsRab21 and (C) OsLEA3 -1 in OsbZIP23 OE lines, RNAi lines and NT plants, where rice polyubiquitin1 (OsUbi1) gene was taken as internal reference. Data bars represent the mean ±SD of triplicate measurement. Statistical analysis by Student’s t-test indicated significant differences (**P<0.01).

Article Snippet: Affinity purified polyclonal antibody raised against OsbZIP23 protein in rabbit (catalog no- E580041-A-SE, EnoGene Biotech, USA) was used as primary antibody (1: 1,000 dilution) and monoclonal plant anti-actin antibody (Sigma, catalog no.-A0480) was used as loading control (1:500 dilution).

Techniques:

Journal: PLoS ONE

Article Title: Enhanced Gene Expression Rather than Natural Polymorphism in Coding Sequence of the OsbZIP23 Determines Drought Tolerance and Yield Improvement in Rice Genotypes

doi: 10.1371/journal.pone.0150763

Figure Lengend Snippet: Germination performance of seeds from (A) OsbZIP23 OE lines (OER#5, OEN#9), and (B) OsbZIP23 RNAi lines (RNAi#1, RNAi#4) in comparison to NT plants on MS agar medium containing 0, 1, 3 and 6 μM ABA at 10 th day. (C) Calculation of the germination rates (%) of OsbZIP23 OE, RNAi and NT seeds. (D and E) Performance of OE, RNAi and NT seedlings in ½ MS liquid medium containing 0, 1, 3 and 6 μM of ABA. Measurement of ( F ) shoot length and (G) root length of OE, RNAi and NT seedlings grown on different concentrations of ABA after 14 days. Data bars represent the mean ±SD of triplicate measurement. Statistical analysis by Student’s t-test indicated significant differences (*P<0.05, ** P<0.01). All the results were based on three independent experiments.

Article Snippet: Affinity purified polyclonal antibody raised against OsbZIP23 protein in rabbit (catalog no- E580041-A-SE, EnoGene Biotech, USA) was used as primary antibody (1: 1,000 dilution) and monoclonal plant anti-actin antibody (Sigma, catalog no.-A0480) was used as loading control (1:500 dilution).

Techniques: Comparison