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Learn to explore and three Bupleurum species differences between the proteome expression profiles of Bupleurum system based on ITS sequences

Author: XieZuo
Tutor: PanShengLi;HuoKeKe
School: Fudan University
Course: Pharmacognosy
Keywords: Bupleurum Radix Bupleuri Herbal Textual Identification and Investigation Medicine Resource Science Phylogeny ITS Sequence Identification Proteome Two-dimensional Electrophoresis Mass Spectrometry
CLC: R282
Type: PhD thesis
Year: 2006
Downloads: 443
Quote: 1
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Abstract


Radix Bupleuri has been frequently used in the prescriptions of Oriental Traditional Medicine in China for two thousand years. Based on the herbal textual identification and investigation, the following proposals have been made. The varieties of Radix Bupleurum embodied in Flora of China should be increased according to the resources of medicinal original plants from genus Bupleurum; The Radix Bupleuri should be classified as the "North", which uses roots as medicine, and the "South" , which uses herb. Consequently, their quality control system should be established respectively; and the artificial cultivation technique of the original plants should be founded to mitigate the stress of feral resources. In this paper, the Medicine Resource Science studies of Radix Bupleuri also have been summarized. It shows that the genus Bupleurum are good pattern plants for the systematic study of biosynthesis of secondary metabolites at the molecular level.The genus Bupleurum (Apiaceae/ Umbelliferae) has around 150 species in the world, and the majority of which are distributed on northern hemisphere. They are particularly abundant in Eurasia and North Africa. In China, 44 species, 17 varieties and 7 forma have been reported, and nearly half of them are native species. In this paper, nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) sequence data were used to investigate the phylogenetic relationships among 70 species (74 taxa) of subfamily Apioideae. Phylogenetic analyses of ITS prove the monophyly of Bupleurum, and support the notion that the Bupleurum should be isolated from subfamily Apioideae and become subfamily Bupleuroideae S. L. Pan. The results also support the subdivision of the Bupleurum into subgenus Penninervia and Bupleurum. Within the subgenus Bupleurum, we propose that four groups could be further divided. And the results indicate that the population of B. falcatum growing in Japan and Korea is different from that in Europe, and should be treated as B. stenophyllum (Nakai) Kitagawa and its variety B. stenophyllum (Nakai) Kitagawa var. kiusianum Kitagawa.In order to find DNA molecular markers for identification, we also studied the nrDNA ITS sequences variation patterns of the medicinal plants of genus Bupleurum in China. The results showed that the homologous alignment of ITS sequences was lower than 75% between genus Bupleurum and outgroups. While within genusBupleurum, it was higher than 87%. The identification standard is >= 99%. Thus ITS sequence is applicable and reliable for identification of Radix Bupleuri at molecular level.In this paper, extraction proteins procedures suitable for two-dimensional electrophoresis (2-DE) from the root materials of different growing periods were established. In particular, an innovative method was established through in-depth study of the perennial flowering period and fruiting period roots, which contain high level of interfering compounds. The remarkable characteristic of our protocol is the proteins distributed in aqueous phase, or chloroform and isoamyl alcohol phase (24/1, V/V), or stayed in the interphase depending on their polarity. Afterwards, the proteins were extracted from these three phases respectively. From interphase, 85% (extracted protein against total proteins) proteins could be extracted and purified. And the phenol extraction is a propriety procedure for extraction proteins from aqueous phase. Based on the well-resolved 2-DE patterns, our protein preparation is free of interfering compounds. And through our method, 3662 spots were separated, and the protein yield was approximately 1.41 mg per 1.0g fresh root material. To our knowledge, this is the first time that a protocol for protein extraction from perennial Bupleurum root has been reported. This protocol appears to have given satisfactory and reproducible results. The protocol is expected to be applied to the study of other recalcitrant plant tissues. Based on this novel method, the proteomic characterization maps of three species Bupleurum have been founded. 94 proteins have been identified from 229 saikosaponins related proteins. The results, which also have never been reported before, can serve as the advantaged basic of the study of saikosaponins and other secondary metabolites biosynthesis.

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CLC: > Medicine, health > Chinese Medicine > Of Pharmacy > Chinese herbal medicines
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