樟叶越桔叶芽总RNA提取方法比较研究

The Comparison on Total RNA Extraction Methods from Vaccinium dunalianum Leaf Buds

本项实验采用改良CTAB、改良SDS、RNApure Plant Kit和Plant RNA Kit 4种方法提取樟叶越桔叶芽总RNA,以期获得适合于樟叶越桔叶芽高质量总RNA的最佳提取方法。总RNA纯度和完整性分别用紫外分光光度计法和琼脂糖凝胶电泳法检测。结果表明,采用改良CTAB和Plant RNA Kit法获得的RNA完整性好,纯度高,A260/A280值分别为2.07和2.08,28S和18S rRNA条带清晰,且前者的亮度约为后者的2倍,无弥散现象。将改良CTAB和Plant RNA Kit法获得的总RNA进行RT-PCR扩增,成功克隆获得了樟叶越桔花色苷5-芳香族酰基转移酶基因长316 bp的cDNA序列。证明改良CTAB和Plant RNA Kit法是樟叶越桔叶芽高质量总RNA的最适提取方法。

In order to obtain optimal extraction method of high quality total RNA from Vaccinium dunalianum leaf buds, improved CTAB, improved SDS, RNApure Plant Kit and Plant RNA Kit methods were used comparatively. The quality of total RNA was examined through OD value and agarose gel electrophoresis. The results showed that using improved CTAB and Plant RNA Kit methods can obtain high purity and quality RNA. The ratios of A260/A280 were 2.07 and 2.08 respectively, the brightness of 28S rRNA was twice that of 18S rRNA, and the total RNA ob- tained was intact and pure. A partial cDNA with 316 bp length of anthocyanin 5-aromatic acyhransferase gene from V. dunalianum was cloned by RT-PCR with RNA as templates obtained by improved CTAB and Plant RNA Kit methods. The results showed that improved CTAB and Plant RNA Kit methods were the better methods to ex-tract high quality total RNA from V. dunalianum leaf buds.