Ed to generate the cDNAs with a RevertAidTM First Strand cDNA

Ed to generate the cDNAs with a RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, Lithuania). The cDNA fragments of CvHsp40, CvHsp70, CvHsc70 and CvHsp90 were obtained by degenerate primers (Table 1) based on the conserved nucleotide sequences of insects which were deposited in GenBank. The gene specific primers of CvHsp40, CvHsp70, CvHsc70 and CvHsp90 (Table 1) were designed for amplifying the full cDNA sequences using a 59-Full Race Kit and 39- Full Race Kit (TaKaRa, Dalian, China) and the full open reading frame (ORF) sequences of CvHsp40, CvHsp70, CvHsc70 and CvHsp90 were verified by PCR. Adult wasp Lixisenatide genomic DNA was isolated using the DNeasy Tissue Kit (Qiagen, Germany), and the introns of CvHsc70 were amplified using ORF-verified primers (Table 1). Amplified fragments were purified using the QIAquick Gel Extraction Kit (Qiagen, Germany) and ligated directly into the pGEM-T Cloning Vector (Promega, Madison, WI). Each fragment-containing plasmid was isolated from cultured E. coli cells by an alkaline miniprep method. Insert fragments were verified by PCR using M13 forward and reverse primers. Sequencing was conducted on an automated fluorescence sequencing system ABI3730 (Applied BioSystems, Foster, CA).Sequence analysisNucleotides and deduced amino acid sequences were analyzed using DNASTAR programs (Version 5.02) (DNASTAR, Inc., Madison, WI, USA). The functional domains and motifs of CvHsps were identified using the programs ScanProsite, Motifscan and SignalP4.0 online (http://www.ca.expasy.org). The obtained amino acid sequences of CvHsps were used to search for homologs in GenBank by BLAST (Position-Specific Iterated-BLAST) software available at the NCBI website (http://www.ncbi.nlm. nih.gov/blast/Blast.cgi). The sequence alignment was performed with Clustal X version 1.81 using default parameters [24] and edited by GeneDoc (Version 2.04) (Free Software Foundation, Inc., MA, USA). The Maximum parsimony (MP) method wasStatistical analysisThe relative transcript amounts of CvHsps were analyzed using one-way analysis of variance (ANOVA). The differences in relative transcript amounts of CvHsps were compared using Dunnett’s multiple comparison and LSD comparison post hoc tests. All statistics were performed using the SPSS software (SPSS 16.0, SPSS Inc., Chicago, IL).Four Heat Shock Protein Genes of Cotesia vestalisFigure 1. Schematic representation of the full cDNAs for CvHsp40, CvHsc70, CvHsp70 and CvHsp90 of Cotesia vestalis. doi:10.1371/journal.pone.0059721.gResults Sequence analysis of the CvHspsCvHsp40. The full length CvHsp40 cDNA (GenBank accession no. JX088376) contains an ORF of 1068 bp encoding a 355 amino acid protein with a predicted molecular weight of 39.1 kDa and theoretical isoelectric point (pI) of 9.12 (Fig. 1 and Fig. S1). Three conserved regions are found in the deduced amino acid sequence of CvHsp40. The first one is a N-terminal J-domain, located at aa 3-57. The second is a glycine/phenylalanine region (G/F domain, aa 70?25). The last region is a C-terminal substrate binding domain (C domain, aa 176?41). Comparing the cDNA and genomic sequences revealed no intron in CvHsp40. Cvhsp70. The full length CvHsp70 cDNA (GenBank accession no. JX088377) contains an ORF of 1938 bp encoding a 645 amino acid protein with a molecular weight of 70.1 kDa and theoretical pI of 5.35 (Fig. 1 and Fig. S2). By Motifscan analysis, we found three conserved characteristic signatures, including IDLGTTYS (aa 6?3), Linolenic acid methyl ester IFDLGGGTFDVSIL (a.Ed to generate the cDNAs with a RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, Lithuania). The cDNA fragments of CvHsp40, CvHsp70, CvHsc70 and CvHsp90 were obtained by degenerate primers (Table 1) based on the conserved nucleotide sequences of insects which were deposited in GenBank. The gene specific primers of CvHsp40, CvHsp70, CvHsc70 and CvHsp90 (Table 1) were designed for amplifying the full cDNA sequences using a 59-Full Race Kit and 39- Full Race Kit (TaKaRa, Dalian, China) and the full open reading frame (ORF) sequences of CvHsp40, CvHsp70, CvHsc70 and CvHsp90 were verified by PCR. Adult wasp genomic DNA was isolated using the DNeasy Tissue Kit (Qiagen, Germany), and the introns of CvHsc70 were amplified using ORF-verified primers (Table 1). Amplified fragments were purified using the QIAquick Gel Extraction Kit (Qiagen, Germany) and ligated directly into the pGEM-T Cloning Vector (Promega, Madison, WI). Each fragment-containing plasmid was isolated from cultured E. coli cells by an alkaline miniprep method. Insert fragments were verified by PCR using M13 forward and reverse primers. Sequencing was conducted on an automated fluorescence sequencing system ABI3730 (Applied BioSystems, Foster, CA).Sequence analysisNucleotides and deduced amino acid sequences were analyzed using DNASTAR programs (Version 5.02) (DNASTAR, Inc., Madison, WI, USA). The functional domains and motifs of CvHsps were identified using the programs ScanProsite, Motifscan and SignalP4.0 online (http://www.ca.expasy.org). The obtained amino acid sequences of CvHsps were used to search for homologs in GenBank by BLAST (Position-Specific Iterated-BLAST) software available at the NCBI website (http://www.ncbi.nlm. nih.gov/blast/Blast.cgi). The sequence alignment was performed with Clustal X version 1.81 using default parameters [24] and edited by GeneDoc (Version 2.04) (Free Software Foundation, Inc., MA, USA). The Maximum parsimony (MP) method wasStatistical analysisThe relative transcript amounts of CvHsps were analyzed using one-way analysis of variance (ANOVA). The differences in relative transcript amounts of CvHsps were compared using Dunnett’s multiple comparison and LSD comparison post hoc tests. All statistics were performed using the SPSS software (SPSS 16.0, SPSS Inc., Chicago, IL).Four Heat Shock Protein Genes of Cotesia vestalisFigure 1. Schematic representation of the full cDNAs for CvHsp40, CvHsc70, CvHsp70 and CvHsp90 of Cotesia vestalis. doi:10.1371/journal.pone.0059721.gResults Sequence analysis of the CvHspsCvHsp40. The full length CvHsp40 cDNA (GenBank accession no. JX088376) contains an ORF of 1068 bp encoding a 355 amino acid protein with a predicted molecular weight of 39.1 kDa and theoretical isoelectric point (pI) of 9.12 (Fig. 1 and Fig. S1). Three conserved regions are found in the deduced amino acid sequence of CvHsp40. The first one is a N-terminal J-domain, located at aa 3-57. The second is a glycine/phenylalanine region (G/F domain, aa 70?25). The last region is a C-terminal substrate binding domain (C domain, aa 176?41). Comparing the cDNA and genomic sequences revealed no intron in CvHsp40. Cvhsp70. The full length CvHsp70 cDNA (GenBank accession no. JX088377) contains an ORF of 1938 bp encoding a 645 amino acid protein with a molecular weight of 70.1 kDa and theoretical pI of 5.35 (Fig. 1 and Fig. S2). By Motifscan analysis, we found three conserved characteristic signatures, including IDLGTTYS (aa 6?3), IFDLGGGTFDVSIL (a.