烤烟烟叶胞嘧啶和5-甲基胞嘧啶分离条件的优化

通过不同流动相、测定温度、流速的对比，优化了分离烟叶总DNA中胞嘧啶和5-甲基胞嘧啶的高效液相色谱法。烤烟烟叶DNA采用CTAB法提取，高氯酸水解，用KOH调节pH到3—5，取上清液测定。色谱柱为NucleosilC18（250mm×4．6mm，5um）。优化后测定条件为：流动相采用甲醇：5mmol／L戊烷磺酸钠（10：90，V／V），流速0．7ml／min，柱温30℃，检测波长273nm，进样量20山。该方法简化了流动相的配制，缩短了分离时间，胞嘧啶和5-甲基胞嘧啶的加标回收率分别为103．50％和96．67％，相对标准偏差为2．35％和2．80％。用该方法研究烤烟烟叶发育过程中总DNA胞嘧啶和5-甲基胞嘧啶的含量，效果良好，符合烟叶生长发育规律。

Optimization of Separating Conditions for Cytosine and 5-Methylcytosine in Flue-cured Tobacco Leaves

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.