论文部分内容阅读
目的:对转Zm C1基因丹参毛状根C1-3株系中8个microRNAs(miRNAs),即miR164b、miR166a、miR166b、miR171a、miR171b、miR172、miR394和miR397进行表达定量分析。方法:以野生型丹参毛状根为对照,采用茎环法(Stem-loop),针对每个miRNA设计3条引物,包括一条特异性反转录引物、一条上游引物和一条通用反向引物。每个miRNA单独反转录,实时荧光定量PCR(q PCR)检测转基因丹参毛状根C1-3中8个miRNA的表达,实验数据用ΔΔCt法进行分析。结果:与野生型丹参毛状根相比,在转基因毛状根C1-3中8个miRNA均表现为不同程度的上调,其中miR394、miR397和miR164最为显著,分别为对照的15.14倍、7.31倍和4.82倍。结论:茎环法可以成功应用于检测丹参中miRNA的表达;C1-3中这几个miRNA转录水平上调可能与Zm C1异源表达引起的有效成分含量及形态发育表型变化相关。
OBJECTIVE: To quantitatively analyze 8 microRNAs (miRNAs), ie, miR164b, miR166a, miR166b, miR171a, miR171b, miR172, miR394 and miR397 in C1-3 transgenic Salvia miltiorrhiza hairy root. Methods: The wild-type Salvia miltiorrhiza hairy roots were used as control. Stem-loop was used to design three primers for each miRNA, including a specific reverse transcription primer, an upstream primer and a universal reverse primer. Each miRNA was reverse transcribed separately and qPCR (qPCR) was used to detect the expression of eight miRNAs in transgenic Salvia miltiorrhiza hairy root C1-3. The experimental data were analyzed by ΔΔCt method. Results: Compared with wild type Salvia miltiorrhiza hairy roots, 8 miRNAs in transgenic hairy root C1-3 showed different degrees of up-regulation, of which miR394, miR397 and miR164 were the most significant, 15.14 times and 7.31 times And 4.82 times. Conclusion: The stem-loop method can be successfully applied to detect miRNA expression in Salvia miltiorrhiza. The up-regulation of these miRNAs in C1-3 may be related to the content of active components induced by heterologous expression of Zm C1 and morphological changes.