DEGseq1. 2. two was implemented to approximately determine the differentially expressed genes by means of the p value as well as the RPKM fold transform worth. The DEGs have been even more studied based upon path way expression analyses and genuine time PCR. Fuel chromatography mass spectrometry profiling The concentrations of ethanol, acetate, alkane, and ter pene during the flower samples have been determined based on gasoline chromatography mass spectrometry, Fresh flower samples have been washed twice with distilled water, subjected to ultrasonic extraction with 10 ml ethyl acetate for 40 minutes, and filtered via a microfiltration membrane, Extracted metabo lites had been analyzed as follows. one ul of sample was injected at a split ratio of 10.1 into a Shimadzu GCMS QP2010 instrument. A VF 5MS capillary column coated with 5% phenyl and 95% dimethylpolysiloxane was employed for separation.
Injection temperature was 230 C as well as the interface temperature was set to 250 C. The ion supply a knockout post was adjusted to 230 C plus the solvent reduce time was set to 3 minutes. Helium was the carrier fuel at a flow price of one. 05 ml minute1. The temperature plan was. an ini tial temperature of 50 C, programmed at 5 C minutes1 to 150 C and held for 10 minutes, then ramped at 10 C minute1 to 260 C and held for 20 minutes. The mass spectrometric detector was operated within the electron im pact ionization mode with an ionizing vitality of 70 eV, scanning from forty 400 m z. Peak identification was per formed by employing AMDIS and WILEY7n databases by using a spectral match high-quality 90%. An in ternal conventional of pentadecanol was added to right for differences in derivatization efficiency and changes in sample volume through heating.
Peaks have been quantified by spot integration and concentrations had been normalized from this source to the quantity on the internal regular recovered. Two technical replicates had been analyzed for three biological samples from each flowering stage. HPLC Profiling The dried flowers were individually comminuted that has a miller. Every strong sample was accurately weighed and extracted with 50 ml of 70% aqueous ethanol by ultrasonication for thirty minutes. The extract was cooled to 25 C and diluted to 50 ml with 70% aqueous ethanol, filtered having a 0. 45 um Millipore filter membrane. Then, ten ul from the filtrate was injected to the HPLC system for evaluation, The HPLC system was an Agilent 1200LC series, consisting of an internet vacuum degasser, a Bin pump SL, an auto sampler, a thermostatic col umn compartment, as well as a diode array detector coupled with an analytical workstation. The column configuration was an Agilent TC C18 reserved phase column, The sample injection volume was 10 ul. The detection wave length was set at 242 nm for evaluation using the flow charge at one. 0 ml minute1, plus the column temperature remaining at 25 C.