柑橘原生质体总RNA提取方法研究

Comparison of different methods of total RNA extraction for protoplasts of Citrus

以悬浮培养的椪柑愈伤组织为材料,通过酶解分离原生质体;采用Trizol法、改良Trizol法、改良CTAB法和TaKaRa RNA提取试剂盒等4种方法提取原生质体的总RNA,通过琼脂糖凝胶电泳、紫外分光光度计和RT-PCR检测其质量和产量。结果表明:4种方法皆能提取出总RNA,其中Trizol法提取的总RNA有降解现象,其他方法所提取的总RNA完整性较好,且D260nm/D280nm和D260nm/D230nm值都在正常范围内;在产量方面,改良Trizol法提取的总RNA产量最高,106个原生质体达43.06μg,分别是改良CTAB法和TaKaRa RNA提取试剂盒的7.74和4.31倍;RT-PCR验证表明,以改良Trizol法提取的RNA为模板所扩增出的目的条带最清晰明亮,且与目的片段大小一致。因此,改良Trizol法是柑橘原生质体总RNA提取的最佳方法。

The protoplasts of callus of Ponkan （Citrus reticulata Blanco） were isolated by the enzymic method and the total RNA of protoplasts were extracted by Trizol method, improved Trizol method, CTAB method and TaKaRa RNA extraction kit. The quality and yield were detected by agarose gel eleetrophoresis, ultraviolet spectrophotometer and RT- PCR. The results showed all four methods can be used to obtain the total RNA. However total RNA degradation was found only in the Trizol method. The other three methods gave good total RNA quality, which were confirmed by the measurements of D260 nm/D280 nm and D26o nm/D230 nm Additionally, the yield of total RNA was the highest in improved Trizol method, up to 43.06 μg· 10-6 protoplasts, which was 7.74 times and 4.31 times of the improved CTAB method and TaKaRa RNA extraction kit, respectively. The result of RT-PCR validation showed that the improved Trizol method gave the most clear and bright band of the PCR product with the expected size. In conclusion, the improved Trizol method is the best way to extract total RNA of citrus protoplasts.