甘蔗原生质体RNA提取条件和方法的优化

为了获得高产量和高纯度的甘蔗原生质体RNA,本研究以新台糖22号（ROC22）和桂糖28号（GT28）甘蔗原生质体为材料,探究了渗透压和原生质体活力对甘蔗原生质体RNA提取效果的影响,并比较了Trizol法、改良Trizol法、改良CTAB法、试剂盒法4种RNA提取方法。结果表明：原生质体渗透压与RNase活性呈显著正相关,原生质体活力与RNase活性呈极显著负相关;RNase活性越高,RNA提取效果越差;使用0.5 mol/L的甘露醇作为渗透压调节剂可获取活力最高的甘蔗原生质体;原生质体活力为70%和90%时所提的RNA完整性好且纯度高,符合后续分子实验的需求,原生质体活力为90%时,RNA产量最高。因此,需要提取甘蔗原生质RNA时,至少需要保证原生质体的活力在70%以上,且原生质体活力越高,RNA的产量越高;采用改良Trizol法,可以大量制备能满足后续分子实验的完整性好且纯度高的RNA,利用该条件和方法,可以确保获得大量高质量的甘蔗原生质体RNA。本研究结果为甘蔗体细胞融合育种过程中的分子生物学研究提供了方法和技术支撑。

Optimization of Extraction Conditions and Methods for Sugarcane Protoplast RNA

In order to obtain high-yield and high-purity sugarcane protoplast RNA, this study investigated the osmotic pressure and protoplast viability on sugarcane protoplasts using the sugarcane protoplasts of ROC22 and GT28, and compared the Trizol method, improved Trizol method, improved CTAB method and kit method. The protoplast osmotic pressure was positively correlated with RNase activity, and the protoplast activity was significantly negatively correlated with RNase activity. Higher RNase activity resulted in a worse RNA extraction effect. Using 0.5 mol/L mannitol as an osmotic pressure regulator, the most active sugarcane protoplasts could be obtained. When the protoplast vigor was 70% and 90%, the RNA integrity was good and the purity was high, which met the requirements of subsequent molecular experiments. The RNA yield was the highest when the protoplast activity was 90%. Therefore, when extracting sugarcane protoplast RNA, the activity of protoplast should be at least above 70%. Using the improved Trizol method, a large number of high-quality RNA could be prepared for subsequent molecular experiments. Using this condition and method, it was ensured that a large amount of high quality sugarcane protoplast RNA was obtained. The results of the study provided methods and technical support for the molecular biology research in the somatic cell fusion breeding of sugarcane.