Heat shock protein 90 (HSP90) a highly conserved molecular chaperone plays

Heat shock protein 90 (HSP90) a highly conserved molecular chaperone plays essential roles in folding keeping structural integrity and regulating the subset of cytosolic proteins. of that at 25 in salinity (‰) for 2?h. Therefore CvHSP90 may be a potential biomarker to monitor environment changes. 1 Introduction Heat shock proteins (HSPs) that first described inDrosophila melanogaster ChondrusPorphyraUndariaLaminariaSaccharinaFucus andUlva[15-22] and a few microalgae such asChlamydomonasandHaematococcus C. vulgarisbecomes a promising candidate bioreactor for large-scale Lenalidomide production of value-added proteins [29].C. vulgarishas important economical and ecological values but often confronts environmental adversities including high temperature and high salinity. Therefore it is often used as a eukaryotic model in studies on stress responses [30]. However the role of HSPs in adverse stress resistance mechanism ofC. vulgarisis yet to be performed. To better understand the mechanism of response byC. vulgaristo different types of environmental stimulation we obtained a HSP90 complementary DNA (cDNA) ofC. vulgarisby combining homology cloning with rapid amplification of cDNA ends (RACE) approaches analyzed in bioinformatics the structural features homologous relationship and phylogenetic position of CvHSP90 and investigated the messenger RNA (mRNA) expression levels of CvHSP90 under different stress conditions using real-time quantitative RT-PCR (qRT-PCR). 2 Materials and Methods 2.1 Sample Collection and Treatment Chlorella vulgariswas presented from Institute of Oceanology Chinese Academy of Sciences and then theChlorellawas grown in Erlenmeyer flasks in F/2 medium that was filter-sterilized through 0.22?Cvulgariswas kept in 40°C for 1?h for extracting total RNA and cloning full-length cDNA of CvHSP90 gene. In Lenalidomide our warmth shock temp treatment Cvulgariswas kept in different temps (5 10 15 20 25 30 35 40 and 45°C) for 1?h to investigate the thermal effect on the manifestation level of CvHSP90 mRNA. In warmth shock time treatment C. vulgariswas kept at 35°C for different times (0?h 1 2 3 4 5 6 7 8 9 10 11 and 12?h resp.) to investigate the effects of warmth shock instances on manifestation level of CvHSP90 mRNA. In salt concentration challenge treatments Cvulgariswas kept at 20°C in different salt concentrations (0 5 Lenalidomide 10 15 20 25 30 35 40 and 45 in ‰) for 2?h to investigate the effects of salt challenges on manifestation level of CvHSP90. 2.2 RNA Extraction Total RNA extraction fromCvulgariswas performed using the TRIzol reagent (Invitrogen). The cDNA first-strand was synthesized Lenalidomide based on M-MLV RT utilization info (Promega) using RQI DNase (Promega)-treated total RNA as template. cDNA blend was diluted to 1 1?:?50 and stored at ?80°C for subsequent fluorescent real-time PCR. 2.3 CvHSP90 cDNA Cloning To amplify the partial fragment of CvHSP90 gene fromChlorella vulgaris= 5) in terms of relative mRNA. Furthermore the data were analyzed by ANOVA (one-way analysis of variance) followed by an unpaired two-tailedt< 0.05. 3 Results 3.1 cDNA Cloning and Sequencing of CvHSP90 Gene A CvHSP90 fragment (745?bp) was amplified by homologous cloning primers P1 and P2 and confirmed highly much like additional known HSP90s. Two pairs of CvHSP90-specific primers (P3-P4 and Lenalidomide P5-P6) that designed in the above sequence were used to clone the full-length cDNA. Rabbit Polyclonal to Synapsin (phospho-Ser9). RACE and nested PCR were performed to amplify the two fragments corresponding to the 3′ and 5′ end of the CvHSP90 cDNA. The full-length cDNA sequence of CvHSP90 was identified 3678?bp by cluster analysis of the above fragments. 3.2 Characterization of CvHSP90 The cDNA sequence of CvHSP90 was submitted in GenBank under accession quantity “type”:”entrez-nucleotide” attrs :”text”:”JQ655149″ term_id :”404276808″ term_text :”JQ655149″JQ655149. The full-length cDNA of CvHSP90 was 3678?bp having a 107?bp 5′ untranslated region (5′UTR) a 1459-bp 3′ untranslated region (3′UTR) having a poly (A) tail and a 2112?bp open reading framework (ORF) encoding a polypeptide that contained 703 amino acids with estimated molecular mass of 80.71?kDa and an estimated isoelectric point of 4.48. The five standard amino acid blocks of HSP90 protein family (NKEIFLRE[L/I]ISN[A/S]SDALDKIR LGTIARSGT IGQFGVGFYSAYLVA[E/D] IKLYVRRVFI G[V/I]VDSEDLPLNISRE) and the consensus MEEVD in the C-terminus were highly conserved as indicated in the CvHSP90 sequence (Number 1) [34]. In the mean time SMART program exposed a typical histidine kinase-like ATPases website in the position 27-182 which is definitely ubiquitous in all HSP90 family members. Figure 1 The full length cDNA sequence of CvHSP90 and its deduced amino acid.