南京师范大学
姓名：芦珊
申请学位级别：硕士
专业：生物学；植物学
指导教师：***
2009-05-10
摘要
元极舞黄草类石斛的基源复杂，在无叶和花的情况下难以鉴别。随着分子生物学的飞速发展，利用DNA进行物种分子鉴别的方法日益成熟，提高了物种鉴别的可靠性、准确性。本研究选取了多种黄草类石斛常见原植物，利用DNA序列，进行了多种石斛的分子鉴别和标记。 利用通用引物通过PCR扩增出了流苏石斛、黄石斛、细茎石斛、细叶石斛、球花石斛、鼓槌石斛、翅萼石斛、黑毛石斛等九种石斛属植物的叶绿体非编码区psb A-trn H片段。实验结果表明，该片段的长度范围在745-768bp，可变位点57个，占整个片段的7.23%，包括15个信息位点，该片段存在5个插入/缺失位点。利用Mega 4.0中的MCL模型计算出了这九种石斛的遗传距离，构建了MP树，分析了它们之间的遗传关系。并提出可将rDNA ITS序列与psb A-trn H间隔区序列联合起来作为石斛属植物DNA条形码。
流苏石斛是药典收录的常用黄草类石斛之一，本研究利用ARMS技术设计了鉴别流苏石斛及其相似种的LSJB ARMS引物，实现了流苏石斛快速准确的分子鉴别。
鉴于翅萼石斛与黑毛石斛不仅外部形态相似，还具有相同的ITS序列，为了建立有效的分子鉴定手段，本实验利用叶绿体DNA的两段非编码间隔区—psb A-trn H及trn T-trn L序列，对这两种石斛进行了分子鉴别，探讨了作为石斛属植物DNA条形码的可行性。实验结果表明，翅萼石斛与黑毛石斛的这两段非编码区序列均具有显著差异，能够达到鉴别的目的；特别是发现了在trn T-trn L片段上的插入/位点处存在叶绿体微卫星（cpSSR），证明了cpSSR是研究种间关系的有效手段之一。
铁橡栎翅萼石斛在我国只分布于云南省，因此对其遗传多样性的研究意义重大。本实验利用磁珠富集法成功开发出了22条翅萼石斛的微卫星，为今后进一步对翅萼石斛遗传多样性研究及保护奠定了基础。
关键词：石斛属，黄草类石斛，分子鉴别，DNA Barcoding，psb A-trn H，trn T-trn L，SSR
The research about molecular maker and authentication of partial species in “Huangcao Shihu”
Abstract
HODGKINHUXLEY模型The species of original plants about “Huangcao Shihu” are multitude and complicated. It is very difficult to identify the Dendrobium species without flowers and leaves. With full speed development of molecular biology, the measure of species identification increasingly mature by DNA sequences, so the reliability and accuracy improved. In this research, we chose the normal species of “Huangcao Shihu” as object, and realized the molecular identification of many Dendrobium species by DNA sequences.
In this paper, we use the universal primers amplify the cpDNA noncoding psb A-trn H fragment of the nine Dendrobium species Dendrobium fimbriatum, Dendrobium tosaense, Dendrobium moniliforme, Dendrobium hancockii, Dendrobium thysiflorum, Dendrobium chrysotoxum, Dendrobium cariniferum, Dendrobium williamsonii. The result showed that the fragment sizes of the psb A-trn H intergenic spacer region varied from 745bp to 768bp. Of the aligned nucleotides 57（7.23%）were variable sites, 15 were parsim-informative. Five indels happened in this region.
We carried out the genetic distance of the nine species by the MCL model in Mega 4.0 and built MP tree, analyzed their genetic relationship. It suggested that it was suitable to combine the ITS and psb A-trn H as DNA barcodes for Dendrobium.
Dendrobium fimbriatum listed in Chinese Pharmacopeia as one commonly used “Huangcao Shihu”. In this paper, we found an effective method to distinguish Dendrobium fimbriatum from its adulterants, ARMS diagnostic primer of Dendrobium fimbriatum has been designed and PCR amplification of Dendrobium fimbriatum was successfully conducted.节油剂
土司文化Dendrobium cariniferum and Dendrobium williamsonii belong to the Sect. Formosae, they have similar appearance and the same nucleotide sequence of ITS. In order to find the effective molecular method to identify them, we adopted two cpDNA noncoding fragments: psb A-trn H and trn T-trn L to identify the two species and approach the feasibility for DNA barcoding.
The result indicated that the two regions existed sequence divergence in the two similar species, which can be used for molecular authentication. Singularity, we found
cpSSR located on the Indels of trn T-trn L region and demonstrated that cpSSR was an effective method in the research about plant species relationship.
Dendrobium cariniferum live only in Yunnan Province in China, so it was greatly significance to research its genetic diversity. In this paper, we developed 22 SSR sequences of Dendrobium cariniferum by magnetic beads hybridization method and estabilish the foundation of Dendrobium cariniferum genetic diversity and protection.
Key words: Dendrobium,“Huangcao Shihu”, molecular authentication, DNA Barcoding, psb A-trn H, trn T-trn L, SSR
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