Molecular authentication and phylogenetic studies of Chinese herbs.

Wang, Yanli.Thesis (M.Phil.)--Chinese University of Hong Kong, 2009.Includes bibliographical references (leaves 90-104).In English with some Chinese characters; abstract also in Chinese.Acknowledgement --- p.IAbstract --- p.III摘要 --- p.VTable of Content --- p.VIIList of Figures --- p.XIIIList of Tables --- p.XVAbbreviations --- p.XVIChapter Chapter 1. --- Introduction --- p.1Chapter 1.1. --- Traditional Chinese Medicine (TCM) --- p.1Chapter 1.2. --- The development history and present situation of Traditional Chinese Medicine --- p.2Chapter 1.3. --- Modernization of Traditional Chinese Medicine --- p.3Chapter 1.4. --- Authentication of Traditional Chinese Medicines --- p.4Chapter 1.5. --- Methods for authentication of Traditional Chinese Medicine --- p.5Chapter 1.5.1. --- Morphological and histological methods --- p.5Chapter 1.5.2. --- Chemical methods --- p.6Chapter 1.5.3. --- Molecular methods --- p.6Chapter 1.6. --- DNA regions suitable for molecular authentication of Traditional Chinese Medicine --- p.8Chapter 1.6.1. --- The chloroplast genome --- p.8Chapter 1.6.2. --- Nuclear sequences --- p.9Chapter 1.6.3. --- Mitochondrial genome --- p.12Chapter 1.7. --- Herb Tu Si Zi --- p.12Chapter 1.7.1. --- The identity of Traditional Chinese Medicine Tu Si Zi --- p.12Chapter 1.7.2. --- The medicinal values of Tu Si Zi --- p.13Chapter 1.7.3. --- Local substitutes of Tu Si Zi --- p.14Chapter 1.7.4. --- The need for molecular authentication of Tu Si Zi --- p.15Chapter 1.8. --- Traditional Chinese Medicinal herbs from Isodon --- p.15Chapter 1.8.1. --- The genus Isodon --- p.15Chapter 1.8.2. --- Xi Huang Cao --- p.16Chapter 1.8.2.1. --- Identity of Xi Huang Cao --- p.16Chapter 1.8.2.2. --- Medicinal values of Xi Huang Cao --- p.17Chapter 1.8.2.3. --- Confusions of herb Xi Huang Cao --- p.17Chapter 1.8.3. --- Dong Ling Cao --- p.18Chapter 1.8.3.1. --- Identity of Dong Ling Cao --- p.18Chapter 1.8.3.2. --- Medicinal values of Dong Ling Cao --- p.18Chapter 1.8.4. --- The molecular authentication of two Isodon herbs --- p.19Chapter 1.9. --- Fagaropsis and Luvunga --- p.20Chapter 1.9.1. --- The classification of Rutaceae --- p.20Chapter 1.9.2. --- Controversial taxonomic issues with Fagaropsis and Luvunga --- p.21Chapter 1.9.3. --- The need of phylogenetic studies of genus Fagaropsis and Luvunga --- p.23Chapter Chapter 2. --- Objectives --- p.24Chapter Chapter 3. --- Materials and Methods --- p.25Chapter 3.1. --- Samples used in this study --- p.25Chapter 3.1.1. --- Tu Si Zi (Dodder seeds) --- p.25Chapter 3.1.2. --- Isodon herbs --- p.28Chapter 3.1.3. --- Fagaropsis and Luvunga --- p.31Chapter 3.2. --- Methods --- p.34Chapter 3.2.1. --- Sample preparation --- p.34Chapter 3.2.2. --- Total DNA extraction --- p.34Chapter 3.2.2.1. --- Cetyltriethylammonium bromide extraction --- p.34Chapter 3.2.2.2. --- Commercial kit extraction --- p.36Chapter 3.2.3. --- DNA amplification --- p.38Chapter 3.2.3.1. --- psbA-trnH intergenic spacer --- p.39Chapter 3.2.3.2. --- trnL-trnF region --- p.39Chapter 3.2.3.3. --- ITS region --- p.42Chapter 3.2.4. --- Agarose gel electrophoresis --- p.43Chapter 3.2.5. --- Purification of PCR product --- p.44Chapter 3.2.6. --- Cloning --- p.46Chapter 3.2.6.1. --- Ligation --- p.46Chapter 3.2.6.2. --- Transformation --- p.46Chapter 3.2.6.3. --- Cell cultivation --- p.47Chapter 3.2.6.4. --- Plasmid extraction --- p.47Chapter 3.2.6.5. --- Insert confirmation --- p.49Chapter 3.2.7. --- DNA sequencing --- p.49Chapter 3.2.7.1. --- Cycle sequencing --- p.49Chapter 3.2.7.2. --- Purification of cycle sequencing product --- p.50Chapter 3.2.7.3. --- DNA analysis --- p.50Chapter 3.2.8. --- Sequence analysis and phylogeny construction --- p.51Chapter Chapter 4. --- Tu Si Zi (Dodder Seeds) - Results and Discussion --- p.52Chapter 4.1. --- Results --- p.52Chapter 4.1.1. --- Dendrogram constructed using psbA-trnH intergenic spacer --- p.52Chapter 4.1.2. --- Dendrogram constructed using trnL-trnF region --- p.53Chapter 4.1.3. --- Dendrogram constructed with the combination of psbA-trnH and trnL-trnF region --- p.59Chapter 4.2 --- Discussion --- p.60Chapter 4.2.1. --- Identification of DNA markers for Cuscuta species --- p.60Chapter 4.2.2. --- Molecular authentication of dodder seeds --- p.60Chapter Chapter 5. --- Isodon herbs - Results and Discussion --- p.64Chapter 5.1. --- Results --- p.64Chapter 5.1.1. --- Dendrogram constructed with internal transcribed spacer 1 --- p.64Chapter 5.1.2. --- Dendrogram established with internal transcribed spacer 2 --- p.65Chapter 5.1.3. --- Dendrogram established with the whole internal transcribed spacer region --- p.66Chapter 5.2. --- Discussion --- p.73Chapter 5.2.1. --- ITS region performing as DNA marker for Dong Ling Cao --- p.73Chapter 5.2.2. --- The identify of TCM materials of Xi Huang Cao --- p.73Chapter Chapter 6. --- Fagaropsis and Luvunga - Results and Discussion --- p.75Chapter 6.1. --- Results --- p.75Chapter 6.1.1. --- Phylogenetic tree constructed with internal transcribed spacer 1 --- p.76Chapter 6.1.2. --- Phylogenetic tree constructed with trnL-trnF region --- p.76Chapter 6.1.3. --- Phylogenetic tree constructed with combined of trnL-trnF region and ITS-1 region --- p.77Chapter 6.1.4. --- The location of Fagaropsis and Luvunga in 3 different phylogenetic trees --- p.78Chapter 6.2. --- Discussion --- p.85Chapter 6.2.1. --- Fagaropsis 一 a member of the ´بProto-Rutaceae´ة group --- p.85Chapter 6.2.2. --- Luvunga 一 a member of Aurantioideae --- p.86Chapter 6.2.3. --- DNA sequencing providing a useful methodology in plant phylogenetic studies --- p.87Chapter Chapter 7. --- Conclusions --- p.89References --- p.90Appendix 1. Sequence alignment ofpsbA-trnH intergenic spacer of dodder --- p.105Appendix 2. Sequence alignment of trnL-trnF region of dodder samples --- p.108Appendix 3. Sequence alignment of ITS region of Isodon herbs and specimens --- p.117Appendix 4. Sequence alignment of ITS-1 region of Rutaceae species --- p.124Appendix 5. Sequence alignment of trnL-trnF region of Rutaceae species --- p.12