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《Shandong Agricultural Sciences》 2010-06
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Optimization of Separating Conditions for Cytosine and 5-Methylcytosine in Flue-cured Tobacco Leaves

JIA Feng1,WEI Qing-hua1,DU Zheng1,WANG Cai-xia1,HUANG Xiao-shu2,LIU Wei-qun3(1.College of Tobacco Science,Henan Agricultural University,Zhengzhou 450002,China;2.Key Open Laboratory of Agricultural Biotechnology and Engineering Technology,Henan Institute of Higher Learning,Zhengzhou 450002,China;3.College of Life Science,Henan Agricultural University,Zhengzhou 450002,China)  
A reversed-phase high performance liquid chromatographic(RP-HPLC) method for the determination of cytosine and 5-methylcytosine in flue-cured tobacco leaves were developed and optimized in mobile phase,detecting temperature and flow rate.The DNA was extracted by CTAB method and hydrolyzed with perchloric acid.Then the pH value was adjusted to between 3 and 5 with KOH.Cytosine and 5-methylcytosine were separated on a reversed-phase C18 column(250 mm×4.6 mm,5 μm),with the mobile phase of methanol+ 5 mmol/L pentane sodium(10︰90,V/V) at a flow rate of 0.7 ml/min.The ultraviolet detector was operated at 273 nm at 30℃.The injection volume was 20 μl.The recoveries of cytosine and 5-methylcytosine in flue-cured tobacco leaves were 103.50% and 96.67% respectively with the relative standard deviation of 2.35% and 2.80% respectively.This method was applied to determine the cytosine and 5-methylcytosine changes in flue-cured tobacco leaves during growth period and the satisfactory results were obtained.
【Fund】: 河南省烟草专卖局科技项目(No.20081225)
【CateGory Index】: S572-34
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