Study On Identification Of Chinese Traditional Medicine Decoction Pieces And Original Plants Using ITS2 Sequence

In this study,the indentification of Taxus plants and its decoction pieces,Scutellaria baicalensis seeds,Sophora flavescens seeds and Caprifoliaceae plants using ITS2 sequence were done to identify Chinese Traditional Medicine Decoction Pieces and Original Plants accurately and quickly.The genomic DNA from samples used study were extracted by improved CTAB and DNeasy Plant Mini Kit.The internal transcribed spacer2 regions in rDNA were amplified and sequenced bi-directionally.Obtained sequences were assembled using the CodonCode Aligner.And the ITS2 sequences were obtained by using the hidden Markov model(HMM)-based annotation methods to remove both ends named 5.8S and 26S(28S).And the secondary structure of the ITS2 sequences were predicted.All the ITS2 sequences were tested by NCBI BLAST,then submitted to GenBank through Sequin and obtained the corresponding accession number.Analyze the sequence through MEGA5.1 to compare the length,the G/C content and the variable sites of the ITS2 sequences,Then genetic distances were computed and the neighbor-joining(NJ)phylogenetic trees were constructed both in accordance with the kimura 2-parameter(K2P)model.Based on the experiment results,the conclusions are as follow:(1)Improved CTAB method is suitable for the DNA extraction of Scutellaria baicalensis seeds and Sophora flavescens seeds,and DNeasy Plant Mini Kit method is suitable for the DNA extraction of Taxus plants and its decoction pieces and Caprifoliaceae plants.The genome DNA extracted by both methods can be used for PCR amplification of ITS2 sequence.(2)The ITS2 sequences can accurately indentify Taxus x media,Taxus cuspidata and Taxus chinensis var.mairei,However it can not distinguish types of Taxus x media.The ITS2 sequences also can indentify Taxus decoction pieces:the content of sample 1 and 2 have Taxus cuspidata;the content of sample 3 have Taxus x media;the content of sample 4 and 5 have Taxus chinensis var.mairei.(3)The experiment also successfully amplify the ITS2 sequences of Scutellaria baicalensis seeds.All the ITS2 sequences of Scutellaria baicalensis seeds purchased from different markets can be divided into two categories:they have four variable sites;the ITS2 sequences of Scutellaria baicalensis seeds purchased from Shanxi,Hebei,Gansu,Neimeng and Henan are the same;the ITS2 sequences of Scutellaria baicalensis seeds purchased from Jiangsu are the same.(4)Analyze 4 ITS2 sequences of Sophora flavescens seeds samples from different origins:the ITS2 sequence of Sophora flavescens seeds are quite conservative and exactly the same.(5)ITS2 sequences can be used to accurately identify Lonicera macranthoides,Lonicera Japonica and Lonicera hypoglauca,but can not be used to indentify types of Lonicera macranthoides.The experiment also successfully amplify the ITS2 sequences of Taxus plants and its decoction pieces,cutellaria baicalensis seeds,Sophora flavescens seeds and Caprifoliaceae plants,and submitted 27 ITS2 sequences to GenBank.ITS2 sequences can be well identified different species,but unable to distinguish types of species,in addition,ITS2 sequences exist variations in Scutellaria baicalensis seeds,but are quite conservative in Sophora flavescens seeds.