F the protein by using the IPEP online proteolysis (http:// ipep.

F the protein by using the IPEP online proteolysis (http:// ipep.moffitt.org/searchProtein.cgi) with parameters of 600?500 Da mass spectrometer and two maximum missed cleavages. Protein abundance was considered significantly up- or down-regulated when the emPAI ratiosLiu et al. Microb Cell Fact (2015) 14:Page 13 ofbetween two medium from two replicates were both higher than 1.25 or lower than 0.8, respectively.Quantitative realtime RTPCR3-hydroxybutyrate dehydrogenase; SHDA: succinate dehydrogenase subunit A; ATPS: ATP synthase subunit beta; IMPDH: inosine-5-monophosphate dehydrogenase; SASPB: small, acid-soluble spore protein B; OAT: ornithine aminotransferase; 3HB: 3-hydroxybutyrate. Authors' contributions XD and LX conceived and designed the study, coordination and helped to draft the manuscript. XL carried out the proteomic studies, and draft the manuscript. YS and SH qRT-PCR analysis. QY, SL, JR and MQ participated in proteomic bioinformatics data analysis. MZ, TW and HH contributed to the bacteria cultures and count experiments. All authors read and approved the final manuscript. Acknowledgements This work was supported by the National Natural Science Foundation of China (31370116), International Cooperation Project (0102011DFA32610), the National Basic Research Program (973) of China (2012CB722301), the National High Technology Research and Development program (863) of China (2011AA10A203), the Key Project of Hunan Provincial Education Department (13CY002, 10CY013, 12K033) and the Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486). Compliance with ethical guidelines Competing interests The authors declare that they have no competing interests. Received: 1 May 2015 Accepted: PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/6833145 11 SeptemberThe relative mRNA levels of selected genes were measured by a two-step real-time RT-PCR analysis with an ABI 7500 Real-Time PCR System (Applied Biosystems, USA) using Power SYBR?Green PCR Master Mix (Applied Biosystems) as previously described [37]. The sequences of the primers used in real-time PCR were developed with Primer version 5.00 (Premier Biosoft International, Palo Alto, CA, USA) and gene-specific primers sequences (Additional file 1: Table S1). For qRTPCR analysis, bacteria were harvested at the time point of 31 h cultivation. The procedures were carried out as described previously. Briefly, the total RNA was isolated using TRIzol Reagent (Invitrogen). The quality and the integrity of the RNA samples were evaluated by absorbance measurements (Thermo Scientific NanoDrop 2000 Spectrophotometers) and agarose electrophoresis (Additional file 1: Fig. S2). Genomic DNA was removed from the total RNA using DNase I (Fermentas) and then the total RNA was retrotranscribed to cDNA using RevertAidTM First Strand cDNA Synthesis Kit (Fermentas) according to the manufacturer's procedure. The cDNAs were used as templates to perform relative quantitative real-time PCR with 16S rRNA as endogenous control. The relative quantification method (delta elta threshold cycle) was used to evaluate quantitative variation between samples examined. mRNA abundance was considered significantly up- or down-regulated when the ratios Capecitabine between two medium were higher than 1.5 or lower than 0.75, respectively.Additional filesAdditional file 1. Figure S1. SDS-PAGE of whole proteins extracted from B. thuringiensis strain. Figure S2. Integrity detection of the RNA samples extracted. Table S1. Primers for quantit.