浙贝母ITS-PCR体系优化与不同产地样品序列差异研究

Optimization of ITS-PCR System of Zhebeimu (Fritillariae Thunbergii Bulbus) and Differences Study of Sample Sequences in Different Producing Areas

目的:优化浙贝母的ITS-PCR扩增体系,并对不同来源浙贝母品种的ITS序列进行差异分析。方法:浙贝母样品采用减压干燥法处理,以广谱植物基因组总DNA提取试剂盒提取浙贝母总DNA,对反应过程中的退火温度、Taq酶浓度、Mg2+、dNTP浓度等因素进行考察。采用Conting Express和DNAman等分子生物学软件进行序列处理。结果:最优的浙贝母ITS-PCR扩增体系为:50μL反应体系中含Taq酶1 U,2.5 mmol/L Mg2+、dNTP分别为4μL,退火温度为56℃。浙贝母的ITS序列全长668 bp,G+C含量为65.4%,不同产地浙贝母的ITS序列无差异。结论:所建立的体系可用以浙贝母ITS序列分析,为浙贝母的ITS序列研究奠定基础。ITS序列不适合作为浙贝母种间和产地间的鉴别工具。

Objective:To optimize the ITS-PCR system of Zhebeimu(Fritillariae Thunbergii Bulbus) and analyze its ITS sequence in different places of origin. Methods:The sample of Zhebeimu was treated by vacuum drying method. The total DNA of Zhebeimu was extracted by the broad-spectrum plant genome total DNA extraction kit. The annealing temperature,Taq concentration,Mg2+and dNTP concentration during the reaction were investigated. Sequence processing was performed using molecular biology software such as Conting Express and DNAman. Results:The optimal ITS-PCR amplification system of Zhebeimu was:50 μL reaction system containing Taq 1 U,both 2.5 mmol/L Mg2+,4 μL dNTP,and annealing temperature:56 ℃. The ITS sequence of Zhebeimu was 668 bp in length and 65.4% in G+ C. There was no difference in ITS sequences among Zhebeimu in different places of origin. Conclusions:The established system can be used to analyze the ITS sequence of Zhebeimu and lay the foundation for the ITS sequence research. The ITS sequence is not suitable as an identification tool for the species and places of origin of Zhebeimu.