is an organism that under certain conditions can produce astaxanthin, an

is an organism that under certain conditions can produce astaxanthin, an economically important carotenoid. believe that astaxanthin accumulation is a defensive reaction of to stresses. Carotenogenesis during stress-induced accumulation of astaxanthin is also well documented by studying the transcriptional expression changes of carotenogenic genes under various stress conditions [6], [7], [8], [10], [12], [13]. Our previous studies revealed that this 5-flanking regions of some key genes involved in astaxanthin biosynthesis, including and remains poorly comprehended [22]. The goals of this study were to investigate the transcriptional expression patterns of carotenogenic genes and accumulating astaxanthin under treatment of exogenous JA in strain 712 was obtained from Institute of Rebastinib Oceanology, Chinese Academy of Sciences. Samples of were produced in MCM medium Rebastinib [24] and cultured in 1000 mL erlenmeyer flasks which were placed in an illuminating incubator (Ningbo Jiangnan Instrument Factory, GXZ-380, Ningbo, China) under a light intensity of 25 mol photons mC2sC1 on a 12 h: 12 h light/dark cycle at 20C without aeration. All flasks were shaken manually twice at fixed time every day. JA Treatment algae in the logarithmic phase were divided into three treatments with 1.25105 cells final content to the control (0 mg/L JA) and two JA treatment groups, JA25 (25 mg/L JA) and JA50 (50 mg/L JA). An equal amount of ethanol was added to the control [17]. After the JA answer was added, the algal cells were harvested at regular intervals over the course of 18 days. Measurement of Astaxanthin Content The astaxanthin was extracted and analyzed following Boussiba and Vonshak [23]. The major absorption peak in dimethylsulfoxide is at ca. 490 nm, and astaxanC thin concentration can be calculated according to formula: C(mg/L)?=?(4.5and represented volume of dimethylsulfoxide and microalgae samples, respectively). Equal aliquots of culture from each treatment and the control were harvested at different time points and lyophilized. Lyophilized cells were then extracted with dimethylsulfoxide repeatedly until the pellet became colorless. Absorbance of the extracts was read at 490 nm with a spectrophotometer (T6 new century, Beijing General Instrument Ltd, China). The blank contained dimethylsulfoxide only. Optical Microscopy Optical microscope observations were made of the morphology, color, biomass and pigment accumulation of and were performed using an optical microscope (Nikon Eclipse 80i microscope, Nikon, Tokyo, Japan). Rebastinib Cell number Cdx1 was counted microscopically using hemocytometer. Rebastinib At least 150 cells and six individual slides were evaluated per sample. RNA Isolation and RT-PCR Samples were frozen in liquid nitrogen and then ground into a fine powder. The total RNA was subsequently extracted using Trizol reagent according to the produces instructions and dissolved in diethypyrocarbonate treated water. In this protocol, RNA was digested with DNaseI. The cDNA used for real-time PCR was synthesized from total RNA using Moloney murine leukemia computer virus reverse transcriptase (Promega Biotech Co., Madison, WI, USA). The gene-specific primers for the eight genes were designed using Primer 3 (Table 1) software and synthesized (BGI, China). The reaction mixture contained 14.5 L pure water, 2 L 10PCR buffer (Mg2+ plus), 0.4 L dNTP (10 mML?1), 1 L (10 ML?1) of each primer, and 0.2 L rTaq DNA Polymerase (TaKaRa, Dalian, China). Amplification was conducted by subjecting the samples to the following conditions: initial denaturation at 94C for 4 min followed by 33 cycles of 94C for 40 s, 55C (? ? accumulating astaxanthin, the growth curves of alga cells, astxanthin concentration, photosynthesis flourescence and the transcriptional expression patterns of eight carotenogenic genes were studied in our experiment. JA-induced Astaxanthin Accumulation in H. Pluvialis Physique 1 shows the growth curves of treatments and control. Algae cells of control grow commonly but decelerate in the later period of incubation. However, growth and breed of samples in treatments were suppressed significantly (p<0.05)..