反相HPLC法研究影响水稻维生素C含量的关键基因

Study on regulation of the key genes on Vitamin C content in the rice by HPLC

HPLC测定水稻叶中维生素C（Vc）含量的测定方法,测定后比较转基因水稻叶与普通水稻叶中Vc含量的差异,为寻找调控水稻中Vc含量的关键基因奠定基础。草酸水溶液提取水稻叶中的Vc后,反相HPLC方法测定含量,选用的色谱柱为Diamonsil C18柱（250×4.6 mm,5μm）,流动相为0.2%磷酸-甲醇,梯度洗脱,检测波长245nm,柱温30℃。测的线性范围为1～40μg/m L（r=0.9992）,平均回收率为98.51%,转基因水稻叶组含量为（0.356±0.009）和（0.444±0.008）mg/g,而普通组Vc含量为（0.554±0.013）mg/g,统计软件SPSS19.0分析表明二者有显著差异（P〈0.01）。此方法测水稻叶中Vc含量具有简便,快速,准确的特点;类受体蛋白激酶439即为影响水稻中Vc含量的关键基因。

The difference of Vc content in leaves was compared between transgenic rice and ordinary rice to find the key genes which regulate the Vc content. Leaf Vc in the rice leaves was extracted with oxalic acid solution. The determination was performed with RPHPLC on Diamonsil C18 column（ 250 × 4. 6 mm,5 μm） under the condition of gradient elution with 0. 2% phosphoric acid-methanol as mobile phase,detection wavelength of 245 nm and column temperature at 30℃. The detection linear range was 1- 40 μg / m L（ r =0. 9992） and average recovery was 98. 51%. The statistic analysis with SPSS19. 0 soft showed significant differences（ P 〈0. 01） of Vc content between transgenic rice leaf group,（ 0. 356 ± 0. 009） and（ 0. 444 ± 0. 008） mg / g,and the normal group,（ 0. 554 ± 0. 013）mg / g. Result showed that this method was simple,rapid and accurate for determination of Vc in rice leaf. The result indicated that receptor protein 439 was the key gene for regulation on Vc content in the rice.