栀子及其近缘茜草科植物、混伪品的rDNA-ITS2序列分析

目的: 利用rDNA-内转录间隔区2(internal transcribed spacer 2,ITS2)序列对栀子及其近缘茜草科植物、混伪品进行快速鉴定。方法: 对研究材料ITS2片段进行聚合酶链式反应(PCR)扩增并双向测序,所得序列经CodonCode Aligner拼接后,序列变异位点采用 PAUP 4.0b10进行统计分析,利用MEGA 5.0软件进行数据分析,并构建邻接(NJ)树,再利用已建立的ITS2数据库及其网站预测ITS2二级结构。结果: 对栀子及其近缘茜草科植物、混伪品ITS2序列长度为201~235 bp,系统发育树上显示不同来源的样本根据亲缘关系的远近各聚一支,表现出单系性;而同时又与混伪品明显分开。比较ITS2二级结构发现,栀子及其近缘茜草科植物、混伪品在4个螺旋区的茎环数目、大小、位置以及螺旋发出时的角度均有明显差异。结论: rDNA-ITS2可以方便快捷地鉴定栀子及其近缘茜草科植物、混伪品。

Objective: To rapidly identify gardenia and relative plants in Rubiaceae as well as its adulterants using DNA barcoding method based on internal transcribed spacer 2(ITS2)sequence.Methods: ITS2 sequence of test samples was amplified by polymerase chain reaction(PCR)and sequenced bidirectionally.Sequence assembly and consensus sequence generation were performed by CodonCode Aligner.The variable sites of the sequence were statistically analyzed by PAUP 4.0b10.The related data analysis was performed using software MEGA 5.0 and the phylogenetic tree was constructed by using the neighbor-joining(NJ)method.The ITS2 secondary structure was predicted using ITS2 web server.Results: The length of ITS2 sequence of gardenia, relative plants in Rubiaceae and its adulterants was 201-235 bp.The samples with close genetic relationship were gathered together on the NJ tress, showing monophyletic, and simultaneously distinguished from their adulterants.The comparison of the ITS2 secondary structure between gardenia, its relative plants in Rubiaceae and its adulterants revealed that gardenia was obviously distinguished from the adulterants in terms of number, size, position of loop, and the angle of helix exsertion.Conclusion: ITS2 barcode could be used to identify Gardenia and relative plants in Rubiaceae and its adulterants effectively.