胡杨根、茎、叶、愈伤组织总RNA提取的研究

Extraction of total RNA from roots,stems,leaves and calli of Populus euphratica Oliv.

胡杨根、茎、叶、愈伤组织等器官和组织的总RNA高质量提取是进行以胡杨RNA为基础的分子生物学研究的基础。以2 a生胡杨实生苗根系为材料,用CTAB-PVP提取缓冲液裂解细胞并变性蛋白,经氯仿/异戊醇(24∶1,v/v)抽提后,用3种不同的沉淀方法获得胡杨根系总RNA,电泳和定量测试等分析提取质量。结果表明,LiCl沉淀法结合70%乙醇洗涤沉淀两次获得的的胡杨根系总RNA,条带清晰、亮度高,且没有明显的DNA污染;actin基因片段设计引物对胡杨根系总RNA进行RT-PCR扩增,扩增片段与预期大小一致,表明该RNA可用于逆转录合成cDNA,提取物RNA中的Li+残留对逆转录酶活性的抑制作用不明显;该方法用以提取2 a生胡杨实生苗茎、叶及愈伤组织总RNA,均获得高质量总RNA。CTAB-PVP-LiCl(附70%乙醇洗涤RNA沉淀2次)的方法可用于胡杨根、茎、叶和愈伤组织总RNA的提取。

The quality of total RNA isolated from roots, sterns, leaves and calli of Populus euphratica Oliv is the foundation of gene expression related researches using the RNA. In this paper, seedling roots of two-year-old P. euphratica were used as test materials, and CTAB-PVP extracting buffer was developed in order to break up organ cells and denaturalize proteins, and three deposition method were used to deposit RNA after the Chloroform/isoamyl alcohol （24： 1, v/v） extraction, then analyzed the RNA quahties by electrophoresis and quantitative test. The result indicated that the bands of isolated RNA were clear, high brightness and without DNA obtained by LiCl deposition and 70%ethanol washing for twice. In addition, a pair of primers was designed to amplify an actin gene of P. euphratica from the root total RNA by RT-PCR, and RT-PCR product was consistent with the expected size, thus this results showed that the root total RNA can be used in Reverse transcription to synthesize cDNA and Li^＋ residues in RNA were too less to restrain the activity of Reverse transcriptase. Moreover, the highquality total RNAs isolated from sterns, leaves and calli of two-year-old P. euphratica were obtained with the CTAB-PVP- LiCl method as well. In conclusion, the CTAB-PVP-LiCl method which was associated with 70% ethanol washing RNA for twice was able to extract total RNA from roots, sterns, leaves and calli of P. euphratica.