Materials and Methods

Crop Hsps Materials and Mehods

Tomato Hsp90
HSP20 Retrieval Gene---Sol Genomics Network, software DNATOOLs, (HMM)
profile- PFam, SMART, Pfam and InterProScan
Sequence Analysis and Structural Characterization--Gene Structure Display
Server 2.0, ProtParam tool, MEME program
Phylogenetic Analysis--- ClustalX 1.83, MEGA 7.0 software- bootstrap test,
Predotar, Wolf Psort, TargetP and MultiLoc, SMART program
Chromosome Localization and Gene Duplication-- software MapDraw V2.1,
PlantGenome Duplication Database (PGDD)
Tissue-Specific Expression Analysis--- Tomato Functional Genomics
Database (TFGD), software MultiExperiment Viewer (MeV)
Expression Profile of SlHsp20 Genes under Different Stress Conditions---
microarray analysis- TFGD, BlastN analysis -database TOM2 oligo
sequences, Affymetrix genome array, Probe Match tool

Plant materials and stress treatments-- Hoaglands solution, NaCl (salt),

20% PEG 6000, liquid nitrogen
RNA isolation, cDNA synthesis and qRT-PCR analysisRNA isolation-Trizol
reagent, NanoDrop 1000 Spectrophotometer, resolving on 1.5%
agarose gel containing 18% formaldehyde, Thermo Scientific Verso
cDNA synthesis kit, StepOne Real-Time PCR Systems, 2Ct
method, Efficiency=10(1/slope) 1 by the default software, MeV4
software
Heterologous expression of SisHSP-27 in yeast and stress tolerance assay--
Yeastmaker Yeast Transformation System
Identification and analysis of genes encoding HSPs (SiHSPs) and phylogenetic
analysis-- HMM profile- HMMER suite, Phytozome, HMMSCAN, CDD
search, ExPASy - ProtParam tool, Gene Structure Display Server v2.0,
MapChart v2.3, MEGA v6.06, ClustalW, NJ method- bootstrap test
Promoter analysis, comparative genome mapping, and duplication and
divergence analysis-- PlantCARE database, BLASTN, Circos v0.55,
MCScanX tool, PAL2NAL
RNA-seq derived expression profiling and methylation analysis of
SiHSP genes--- European Nucleotide Archive, MeV v4.9, Illumina
Genome Analyzer (GAIIx), Bismark tool

C4 model- HSP100, 90,

foxtail millet 70, 60, sHSP

Genome-wide identification of HSP70 proteins in soybean--

Phytozome v9.1, HMM profile- Pfam database, BLASTP- soybean
genome database, SMART tools, pI/Mw tool-ExPASy
Subcellular localization and promoter analysis of the GMHSP70
family-- WoLF PSORT& TargetP 1.1, plantCARE database

Soybean Multiple alignment and phylogenetic analysis-- TAIR & TIGR,

(Glycinemax L.) HSP70 Clustal X2.0 software, MEGA 5.0-NJ method, bootstrap analysis
 Gene structure analysis and identification of conserved motifs--
Gene Structure Display Server (GSDS), MEME

Chromosomal location and gene duplication-- MapChart software,

SoyBase browser, Smith-Waterman algorithm

RNA-seq atlas analysis-- RNA Seq-Atlas, MeV v4.8 software

Plant growth and stress treatments-- PEG3000, liquid N2

RNA extraction and qRT-PCR analysis-- Trizol reagent, DNaseI,

TransScript First-Strand cDNA Synthesis SuperMix Kit, Primer 5.0,
StepOne Real-Time PCR System, 2CT method

Hsp20
Database screening and sequence analyses-- The soybean genome
annotation database & Phytozome v8.0, Blast searches-HMM- PFam,
Phytozome database, MEME, InterProScan & PROSITE, EXPASY
PROTPARAM, PlantCare- PLACE- MEME/TOMTOM & MatInspector,
Genevestigator, Soybase, LGE - Soybean Genome Project, MapChart
software, SignalP program, Predotar & TargetP, WoLF PSORT program
, MitoProt II - v1.101 or PTS1 predictor, TMHMM 2.0 program, ClustalX
2.1, NJ method- MEGA 5 software, Phyre

Growth conditions and stress treatments-- Embrapa Soja Active

Germplasm Bank, liquid nitrogen,

plant RNA isolation-- RNA extraction kit with the TRIzol reagent,
Uniscience NanoDrop ND-1000 spectrophotometer, agarose gel
electrophoreses, deoxyribonuclease I (Kit DNaseI, Invitrogen),
SuperScript III Kit,

qRT-PCR-- qRT-PCR, software Primer3Plus & Vector NTI Advance,

1X SYBR Green Master Mix Kit, qRT-PCR thermocycler, Sequence
Detection program, REST software version 2.0.7

Screening for putative TFBS (transcription factor binding site)

combinatorial models-- FrameWorker Genomatix suite of programs

Database searches, identification and gene classification of DnaJ

family genes in soybean--soybean (Glycine max Wm82.a2.v1)
database -HMM file, Phytozome, ExPASy online software, NCBI
Conserved Domain Search program, SMART tools & and Pfam database

Multiple alignment and phylogenetic analysis--ClustalX 2.0

software, MEGA 5.0 software- ML method

DnaJ/HSP40 Exon/intron organization, chromosomal location and gene

 duplication--Gene Structure Display Server, MapInspect software

Microarray analysis-- NCBI Gene Expression Omnibus database,

MATLAB software

Plant materials, growth conditions, and stress treatments--98%

sulfuric acid, Hoagland solution, 50 mM NaHCO3, liquid nitrogen, TAIR,
PCR and semi-quantitative RT-PCR, 5% NaClO, MS solid medium

Quantitative real-time PCR--EasyPure Plant RNA Kit protocol,

SuperScriptTM III Reverse Transcriptase kit, SYBR Green Master Mix,
2CT method

Vector construction and generation of transgenic Arabidopsis

thaliana--USER cloning method, floral dip method, phosphinothricin
ammonium, semi-quantitave RT-PCR

Phenotypic analysis of Arabidopsis plants

Statistical analysis--Duncans multiple range tests or Students t test

Data sources--Rice Genome Annotation Project (RGAP version 6.0),

Database of Interaction Proteins (DIPs ), GEO , Affymetrix GeneChip
Rice Genome Array
Microarray analysis--impute package (version 1.22.0) in
Bioconductor , robust scatterplot smoother (LOWESS) in R
software, Limma package (version 3.2.0), Boxplot in R, bootstrap
method
Identification of rice Hsps--Uniprot database, MSU Rice Genome
Annotation Project, Ricechip.org
Prediction of proteins interacting with Hsp70s in rice--BLASTP
(version 2.2.23+), t-test
Assessment of PPIs of Hsp70s in rice-- WoLF PSORT, agriGO
Identification of binding sites of Hsp70s in rice-- rice proteome, Pfam ,
PfamScan program (version 091007) & HMMER package (version 3.0b3),
PROSITE , ScanProsite tool, Fishers exact test, R software, BH method
Hsp70 network in rice-- GO database, Cytoscape, LAMP (Linux, Apache,
MySQL and PHP) platform, Open Flash Chart & Google Chart Tools

HSP70(case
Rice study)