System lightcycler 480 equipment

ATP phosphoribosyltransferase (HisG), which catalyzes the first step of histidine biosynthesis, is the most important enzyme regulated at the enzymatic level. Therefore, in this study, hisG gene expression was analyzed to understand the synthesis state of L-histidine. DNA from the culture medium was isolated from 0.5 g using nucleic acid reagent (Takara accompany, Dalian, China) following the manufacturer’s instructions. DNA quality and concentration were tested by gel electrophoresis and a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, United States). The hisG primers were obtained according to a previous paper (18 S FWD: GTGCCAGCAGCCGCGGTA; 18 S RV: TGGACCGGCCAGCCAAGC; Huada Gene Technology Company, Shenzhen, China). Quantitative RT-PCR (qRT-PCR) was carried out in a 10-μL reaction vessel containing 5 μL 2.5 × RealMaster Mix, 20 × SYBR solution (Takara accompany, Dalian, China), 0.2 μL of both forward and reverse primers, and 1 μL of diluted cDNA (1:10). PCR amplification was performed using System LightCycler 480 equipment (Roche Applied Science, Germany); the qRT-PCR procedure comprised 95°C for 1 min, followed by 40 cycles of 95°C for 10 s, 55°C for 30 s, and 68°C for 1 min. Values for gene expression were calculated following the method outlined by Rieu and Powers (2009) (link) using delta-delta Ct.

Cold-regulated genes were identified in this study using the National Center for Biotechnology Information (NCBI) database (www.ncbi.nlm.nih.gov), while primers for RT-PCR genes were designed and amplified using the Primer 3 online tool (www.primer3). Quantitative RT-PCR (qRT-PCR) was carried out in a 10 μl reaction vessel containing 5 μl 2.5 × RealMaster Mix, 20 × SYBR solution (Tiangen Biotech, Beijing), 0.2 μl of both forward and reverse primers, and 1 μl of diluted cDNA (1:10). PCR amplification was performed using System LightCycler 480 equipment (Roche Applied Science, Germany); our qRT-PCR procedure comprised 95°C for 1 min, followed by 40 cycles of 95°C for 10 s, 55°C for 30 s, and 68°C for 1 min. Values for gene expression were calculated following the method outlined by Ramakers et al. (2003 (link)) using delta-delta Ct; all the gene primers used for RT-PCR in this study are listed in Table 3.