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目的:对甘草道地性形成机制进行探索。方法:在GenBank中检索β-香树酯醇合成酶(β-AS)基因,进行序列比对并找出序列保守区,用primer premier 5.0软件设计引物,对3个不同产地甘草材料的β-AS基因进行扩增、测序,用MegAlign软件进行序列分析并构建系统树。在表达差异实验中,对来自3个产地的9份甘草材料进行总RNA提取,逆转录得到cDNA,以甘草18S基因为内参,PCR扩增后进行相对表达量分析。结果:在序列多态性实验中,9个样品的β-AS序列经比对分析共发现108处变异位点,内含子变异位点数高于外显子变异位点数两倍。在表达差异实验中内蒙组、甘肃组和宁夏组的甘草β-AS基因平均相对表达量有显著性差异。结论:不同产地甘草β-AS基因多态性的差异以及表达量的差异,可能是导致甘草道地性形成的原因之一。
Objective: To explore the mechanism of the licorice formation. Methods: The β-AS gene was retrieved from GenBank and sequenced. The conserved region of the sequence was identified. Primers were designed with primer premier 5.0 software. The β- AS gene was amplified, sequenced and sequenced with the MegAlign software and a phylogenetic tree was constructed. In the expression difference experiment, total RNA was extracted from 9 parts of licorice from three producing areas, and the cDNA was reverse transcribed. The relative expression amount of 18S gene of licorice root was used as an internal control after PCR amplification. Results: In the sequence polymorphism experiment, the total of 108 polymorphic sites were found in the β-AS sequences of 9 samples, and the number of introns was more than twice that of exon. In the expression difference experiment, the average relative expression level of β-AS gene in Inner Mongolia, Gansu and Ningxia groups was significantly different. Conclusion: The differences in β-AS gene polymorphism and the differences in expression levels of Glycyrrhiza uralensis in different areas may be one of the reasons leading to the licorice formation.