胡枝子高质量RNA的提取及ALMT基因片段的克隆和表达分析

摘要

The total RNA from the root and shoot of woody plant Lespedeza bicolor, which had plenty of polysaccharides and polyphenols were extracted by improved CTAB method. The values of OD260/OD280 of the extracted RNA were between 1.9-2.1, the yields were between 100-250 μg/g FW. ALMT gene fragment was successfully amplified from cDNAs reverse-transcribed from the total RNA and its expression was analyzed by semi-quantitative RT-PCR and quantitative real-time RT-PCR. The sequence of the cloned ALMT gene fragment in Lespedeza bicolor was highly identical to other species' ALMT gene sequences. This gene was Al-inducing and mainly expressed in roots. These results showed that the total RNA extracted from Lespedeza bicolor by this method was of high quality, and could be used for the following study on gene expression.%针对木本植物胡枝子组织中多糖、多酚等次生物质含量高的特点,采用改良的CTAB法,成功地从胡枝子组织中提取了总RNA。所提取的胡枝子总RNA OD260/OD280值介于1.9～2.1之间,产率介于100～250μg/g之间。采用该方法提取的胡枝子总RNA,经过RT-PCR成功地克隆到了胡枝子ALMT基因片段,克隆得到的胡枝子ALMT基因片段序列与已知ALMT基因序列之间具有较高同源性,经半定量RT-PCR和实时荧光定量PCR表达分析,证明该基因在根中表达且受铝诱导。试验结果表明利用改良的CTAB法提取的胡枝子总RNA样品质量较高,能够用于基因的半定量和定量表达分析。