大叶茶叶绿体基因组从头测序
胡娜
学位类型硕士
导师高立志
2010-11
学位授予单位中国科学院研究生院
学位专业植物学
摘要大叶茶(Camellia sinensis var. assamica)隶属于山茶属(Camellia)之茶组(Section Thea)。因其含有多种药用和保健成份,诸如儿茶素类、茶多酚和咖啡碱等而广泛地受到人们的关注。叶绿体因基因组小,编码区及非编码区进化速率的差异而越来越多地被用于各类分类阶元的系统发育研究;而且,利用叶绿体基因工程来改良作物的产量及重要化合物的含量具有重要意义。然而,目前茶组植物叶绿体基因组DNA的提取因为其叶片的特殊结构和成分而困难重重。本研究根据茶组植物的特殊性质,优化各个提取参数,建立了一套适用于茶组乃至山茶属植物的叶绿体DNA提取方法。利用此方法提取的叶绿体DNA和Solexa测序仪,首次获得了第一个山茶属植物,大叶茶(C. sinensis var. assamica)的叶绿体全基因组。此外,本论文还利用藻类基部类群的叶绿体全基因组重组信息重建其系统发育关系,探讨了利用基因组重组信息重建系统发育的可行性。主要研究结果如下:1. 茶组植物叶绿体 DNA 的提取, 根据茶组植物叶片的性质,优化各个提取参数,得到了适用于茶组植物叶绿体 DNA 提取的条件:用短时高速匀浆进行细胞的破碎,同时用低 pH 的提取液辅助匀浆;用差速离心法获得完整的叶绿体;用高盐缓冲液去除破碎细胞的核DNA 残留;得到纯度高,浓度大且酶切成功的叶绿体总 DNA ,该总 DNA 可以直接用于二代测序仪 Solexa 的制样测序。2. 大叶茶叶绿体全基因的获得, 利用二代测序仪 Solexa,获得了茶组,同时也是杜鹃花目的第一个叶绿体全基因组—大叶茶的叶绿体全基因组序列。其基因组全长为157,140bp,其中LSC区长度为86,600bp,SSC区长度为18,272bp,IR区长度为26,134bp,共含有基因115个,其中包括30个tRNA基因,4个rRNA基因。利用得到的大叶茶的叶绿体基因组序列,比较了包括其在内的菊分支的三个喜阴植物和四个阳生植物的叶绿体基因组,发现两者的结构变异很小,但是阳生植物的大部分基因的核苷酸替换速率大于喜阴植物。推测可能是阳生植物叶片细胞中有更多的叶绿体,积累了更多的突变造成的。3. 基于叶绿体基因组重组重建系统发育关系的可行性研究—藻类基部类群的系统发育重建, 首次根据三个藻类基部类群,灰藻门(glaucophyta),绿藻门(chlorophyta)和红藻门(rhodophyta)的叶绿体全基因组的重组信息,通过共同基因含量、保守片段的重组以及基于基因顺序的信息得到了它们的系统发育树。与绿藻门相比,灰藻门与红藻门含有更多的共同基因;灰藻门中的保守基因串与红藻门具有更大的相似性;系统发育树也支持灰藻门与红藻门形成单系,而与绿藻门构成姐妹类群。
资助项目C. sinensis var. assamica is an important member of section Thea, belonging to the genus Camellia. It becomes more and more attractive because of its medicine and health maintenance efficacy. The chloroplast genome sequence is now widely used to resolve the phylogeny at different levels of taxonomic categories, because of its small genome size and different evolutionary rates of coding and noncoding gene regions. Futhermore with the progression in chloroplast genetic engineering, we can improve the quantity and quality of crops efficiently. However, the isolation of high yield and purified chloroplast DNA of Camellia becomes very difficult due to special leaf structure and complicated metabolic products. Unfortunately there are not any efficient methods available to isolate the Camellia cpDNA. In this study, we established an efficient method to isolate the Camellia cpDNA on basis of its leaf structure and complicate compounds. With the purified and sufficient cpDNAs, we completed sequencing the first chloroplast genome in the Genus Camellia, even the first one in the order Ericales. Besides, we used the information of genomic rearrangement of chloroplast to reconstruction the phylogenetic relationship of the three basal algal groups to investigate the feasibility of using genomic rearrangement for phylogeny reconstruction. Results are as below: 1. Isolation of cpDNAs of Section Thea of the genus Camellia, Based on special leaf structure and components of Section Thea of the genus Camellia, we adjusted experimental conditions and established a suitable protocol for its chloroplast isolation. They included short time and high speed homogenate, combined with a low pH extraction buffer for cell breakage, differential centrifugation for isolating the chloroplasts, high-salt wash buffers for the elimination of nuclear DNA contamination and phenol-chloroform treatment. As a result, sufficiently purified and successfully digested cpDNAs was abtained. They can be directly sequenced by Solexa. 2. Complete chloroplast genome sequence of C. sinensis var. assamica, With the second generation sequencing technology of Solexa, we completed the first chloroplast genome sequencing of the genus Camellia, also the first one of order Ericales. The genome size is 157,140bp, and the lengths of LSC, SSC, IR are 86,600bp, 18,272bp, 26,134bp, respectively. The genome contains 115 genes, including 30 tRNA genes and 4 rRNA genes. Futhermore, with the complete cpDNA of C. sinensis var.assamica and other completely sequenced cpDNAs of Asterids, we compared shade-loving and sun plants, the chloroplast structures of the two kinds of plants are very similar, but we found that nucleotide substitution rates of shade-loving plants are lower than sun plants. A likely explanation is that more variations accumulated by more chloroplasts in cells of sun plants. 3. Feasibility to use genomic rearrangement for the phylogeny reconstruction: a case study on the phylogeny reconstruction of basal alagal groups, Phylogeny of three basal algal groups, glaucophyta, chlorophyta and rhodophyta, was first reconstructed by means of rearrangement information from the chloroplast genomes. It includes common genes content, conserved gene clusters and phylogenetic trees produced by gene order data. We found that glaucophyta and rhodophyta share more common genes, and the conserved gene clusters between them have fewer arrangements. Furthermore phylogenetic trees also showed closer relationships between glaucophyta and rhodophyta. Thus our results supported that chlorophyta are sister to glaucophyta plus rhodophyta.
语种中文
文献类型学位论文
条目标识符http://ir.kib.ac.cn/handle/151853/16180
专题昆明植物所硕博研究生毕业学位论文
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胡娜. 大叶茶叶绿体基因组从头测序[D]. 中国科学院研究生院,2010.
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