基于叶绿体rbcL基因对不同产区浙贝母的PCR扩增体系及序列差异研究

目的:建立不同产区浙贝母的叶绿体rbcL基因的PCR扩增体系,并对产区间的序列差异进行分析。方法:将采集于27处不同产地的浙贝母鉴定、切碎、打粉后保存备用。总DNA提取方法采用试剂盒法,PCR扩增体系主要考察退火温度、Taq酶浓度、Mg^2+、dNTP浓度等因素。基因序列分析采用Conting Express和DNAman软件。结果:50μL反应体系中含Taq酶1 U,2.5 mmol/L Mg^2+、dNTP分别为2μL,退火温度为52℃。浙贝母rbcL序列全长599 bp,G+C含量为50.7%,浙贝母不同产区rbcL序列未发现突变位点。结论:所建立的体系可用于浙贝母rbcL序列分析,为浙贝母的rbcL序列研究奠定基础。研究结果提示rbcL序列在浙贝母种间或者产地间的分辨率较低,不适合作为浙贝母种间和产地间的鉴别方法。

Study on PCR Amplification System and Sequence Difference of Zhebeimu(Fritillariae Thunbergii Bulbus) in Different Producing Areas Based on Chloroplast rbcL Gene

Objective:To establish a PCR amplification system for the chloroplast rbcL gene of of Zhebeimu(Fritillariae Thunbergii Bulbus) in different producing areas,and to analyze the sequence differences in the different producing areas. Methods:Zhebeimu collected in total 27 different producing areas were identified,chopped,powdered and stored for later use. The total DNA extraction method uses the kit method. The PCR amplification system mainly investigates the annealing temperature,Taq enzyme concentration,Mg~(2+),dNTP concentration and other factors. Gene sequence analysis was performed using Conting Express and DNAman software. Results:The 50 μL reaction system con tained 1 U of Taq enzyme,2.5 mmol/L Mg~(2+) and 2 μL dNTP respectively,and the annealing temperature was 52 ℃. The rbcL sequence of Zhebeimu was 599 bp in length and 50.7% in G+ C. No mutation sites were found in the rbcL sequences of Zhebeimu in different regions. Conclusions:The established system can be used for the analysis of rbcL sequence of Zhebeimu,which lays a foundation for the study of rbcL sequence of Zhebeimu. The results suggest that the rbcL sequence has a low resolution between the different species of Zhebeimu or between the different producing areas;therefore,it is not suitable to be used as a method for identification between different species and producing areas of Zhebeimu.