Subcellular location of TaHsp90 as predicted by CELLO v.2.5 server.

<p>Subcellular location of TaHsp90 as predicted by CELLO v.2.5 server.</p

List of TaHsp90 genes retrieved from NCBI database for analyzing nucleotides and amino acid sequences.

<p>List of TaHsp90 genes retrieved from NCBI database for analyzing nucleotides and amino acid sequences.</p

Heat stress transcripts, differential expression, and profiling of heat stress tolerant gene <i>TaHsp90</i> in Indian wheat (<i>Triticum aestivum</i> L.) cv C306

<div><p>To generate a genetic resource of heat stress responsive genes/ESTs, suppression subtractive hybridization (SSH) library was constructed in a heat and drought stress tolerant Indian bread wheat cultivar C306. Ninety three days old plants during grain filling stage were subjected to heat stress at an elevated temperature of 37°C and 42°C for different time intervals (30 min, 1h, 2h, 4h, and 6h). Two subtractive cDNA libraries were prepared with RNA isolated from leaf samples at 37°C and 42°C heat stress. The ESTs obtained were reconfirmed by reverse northern dot blot hybridization. A total of 175 contigs and 403 singlets were obtained from 1728 ESTs by gene ontology analysis. Differential expression under heat stress was validated for a few selected genes (10) by qRT-PCR. A transcript showing homology to <i>Hsp90</i> was observed to be upregulated (7.6 fold) under heat stress in cv. C306. CDS of <i>TaHsp90</i> (Accession no. MF383197) was isolated from cv. C306 and characterized. Heterologous expression of <i>TaHsp90</i> was validated in <i>E</i>. <i>coli</i> BL21 and confirmed by protein gel blot and MALDI-TOF analysis. Computational based analysis was carried out to understand the molecular functioning of TaHsp90. The heat stress responsive SSH library developed led to identification of a number of heat responsive genes/ESTs, which can be utilized for unravelling the heat tolerance mechanism in wheat. Gene <i>TaHsp90</i> isolated and characterized in the present study can be utilized for developing heat tolerant transgenic crops.</p></div

Gene expression analysis by qRT-PCR in contrasting wheat cultivars C306 (heat tolerant) and HD2967 (heat susceptible).

<p>(A) Fold change expression of Hsp90, <i>Hsp-Sti</i>, hypothetical <i>Dnaj</i>, ClpB1/Hsp100, PPIase, <i>GAPDH</i>, AAA ATPase, FKBP, PSBR, hypothetical <i>Hsp</i> at 37°C. (B) Fold change expression at 42°C. (C) Fold change expression of <i>Hsp90</i> in cv. C306. (D) Fold change expression of <i>Hsp-Sti</i> in wheat cv. C306. (E) Fold change expression of hyp <i>Dnaj</i> in wheat cv. C306. Statistical significance has been shown by asterisk (*) at p≤0.05 (n = 3).</p

Evolutionay relationship of <i>T</i>. <i>aestivum</i> cv. C306 <i>TaHsp90</i> with other plant species.

<p>(A) Based on the nucleotide sequence. (B) Based on the amino acid sequence. Evolutionary analyses were conducted in MEGA7 using Neighbour-joining method with boot strap value of 1500.</p

3D structure prediction of TaHsp90.

<p>(A) Modelling of TaHsp90 based on template-c5fwkA. (B) TaHsp90 docking to a predicted ligand (colored portion shows active site interaction with ligand). (C) Ramachandran plot showing the favourable region of TaHsp90 protein.</p

TaHsp90 phosphorylated sites and secondary structure prediction.

<p>(A) Phosphorylated sites prediction in TaHsp90 using Netphos 3.1a server. (B) Secondary structure map of TaHsp90 shows helices, sheets and disorderness. (C) Amino acid composition percentage along with the predicted significance p value. Feature predictions are color coded according to the sequence feature key shown below.</p

Potential N-linked glycosylated sites found in TaHsp90.

<p>The result was generated using NetNGlyc 1.2 server.</p

Tissue specific digital expression analysis of TaHsp90 with closely related genes.

<p>(A) Digital expression analysis of genes closely related to TaHsp90. (B) Pearson correlation coefficient analysis of TaHsp90 with closely related genes. (C) String based analysis of TaHsp90 for protein to protein interaction.</p

TaHsp90 digital expression in wheat.

<p>(A) TaHsp90 expression at 10 different stages of development (B) TaHsp90 expression at 26 anatomical parts of wheat tissue, key color code features given and # represents the number of samples in which the expression was observed. IQR stands for Interquartile range.</p