中国科学院昆明动物研究所机构知识库

烟草属为茄科中的第五大属，包含75种自然生长的物种。烟草属植物为一年生或多年生，原产于美洲、大洋洲和南太平洋的一些岛屿，是最早应用于分子生物和基因工程研究的模式植物，具有很高的科学价值和农业价值。烟碱是烟草属植物所能产生的一种特有生物碱，具有抵抗病原体和食草动物的功能，同时也扮演着多种生理学角色。尽管目前已有一些关于烟草属植物进化关系的研究，但它们大都仅使用了少量基因，例如，nrDNA、质粒DNA、ncpGS等，因而很多节点的bootstrap值都很差。此外，关于烟碱生物合成和代谢方面的研究大都也只关注了少量物种的少量基因。随着二代测序技术的进步，在获取烟草属物种基因组方面已经取得了较大进步。尽管目前已有7个烟草属植物的基因组序列被释放，然而这对烟草属的遗传研究仍不足够。相较于全基因组测序，RNA测序为探究那些没有参考基因组序列的物种提供了机会。该技术耗费小且更易于处理，但仍可以产生相对丰富的信息，已经成功应用于烟草、拟南芥、苜蓿和大豆等多个物种的研究。本论文选取了来自8个烟草组中的16个烟草属物种进行研究。对烟草叶片转录组进行了de novo组装，并根据NR、Swiss-Prot、KEGG和GO四个数据库进行基因注释。基于转录组数据，我们进行了烟草属物种的进化分析、基因表达量和烟碱含量相关性分析、WGCNA分析和重要基因分析，主要结果如下：(1) 共识别了2,167单拷贝orthologs，使用最大似然法和邻接法，构建出拓扑结构相似的进化树，相较于以往基于形态学或少量基因构建的进化树，本论文构建的关于16种烟草属物种的进化树更为稳健，不同物种间的进化关系也更为可靠。(2) 发现Tomentosae组中的N. otophora与Nicotiana组中的N. tabacum紧邻，可能是由于N. tomentosiformis和N. otophora的杂交种是N. tabacum的父系，因而N. otophora在N. tabacum的形成过程中有附加的贡献。Alatae组中染色体数目不同的N. bonariensis和N. longiflora却更为接近，与以往基于质粒DNA序列的研究结果不一样，需要进一步研究。(3) 获得了16个烟草属物种的转录表达谱，分析了候选基因的表达量与烟碱含量的相关性，发现与烟碱合成有关的候选基因的表达量与总烟碱在整体上成正相关，而与烟碱降解有关的候选基因的表达量与有效烟碱则在整体上成负相关。(4) 识别了一些与烟碱含量强相关的基因：喹啉酸磷酸核糖转移酶QPT2、天冬氨酸氧化酶AO、小檗碱桥接酶BBL和N-腐胺甲基氧化酶MPO。相反地，N-腐胺甲基转移酶PMT和异黄酮还原酶A622在所有烟草属物种中的表达量都极低，意味着这两步在叶片中并不活跃。(5) 检测了KNOX、抗冻、N和NBS基因在16个烟草属物种中的含量，发现N. pauciflora和N. undulata中KNOX含量最高，N. undulata中抗冻基因含量最高，N. bonariensis中N和NBS基因含量最高。此外，在博士学习期间，还利用细胞质基因组，进行了两个重要保护物种的遗传研究。一个是我国特有的原羚属珍稀物种普氏原羚（Procapra przewalskii），其已被列入世界自然保护联盟濒危物种红色名录，另一个是2011年刚被发现和命名的深圳拟兰（Apostasia shenzhenica），国际上所有野生兰科植物均已被列入濒危野生动植物种国际贸易公约。在分子生物学和基因组时代，细胞质基因组为动植物分类、系统发育和物种鉴定等提供了不可或缺的遗传信息。本论文测定了普氏原羚完整的线粒体基因组，取用了深圳拟兰叶绿体基因组数据，基于普氏原羚和其它牛科物种的线粒体基因组以及深圳拟兰与其它兰科物种的叶绿体基因组，分别开展了牛科和兰科物种的进化分析，主要结果如下：(1) 建立的牛科物种和兰科物种进化树均具有较高的bootstrap支撑。(2) 发现Procapra属的P. przewalskii与P. gutturosa接近，而P. gutturosa与Madoqua属物种亲缘关系并非最近。(3) 发现Apostasioideae亚科的A. shenzhenica与A. odorata相近，而Apostasioideae和Cypripedioideae两个亚科聚集在不同的进化分支中。

Nicotiana is the fifth-largest genus in Solanaceae, contains 75 naturally grew species. Plants in Nicotiana genus are annual or perennial, native to America, Oceania and some islands of South Pacific. They are the model plants first applied to molecular biology and genetic engineering studies, and have high scientific and agricultural values. Nicotine is a specific alkaloid of Nicotiana species, and functions in defense against pathogens and herbivores as well as serving diverse physiological roles.Although there had been several studies on the phylogenetic relationships in the Nicotiana genus, most of them were only used a few genes, such as nrDNA, plastid DNA and ncpGS, and thus many nodes had poor bootstrap supports. Besides, most of the genes related to the nicotine pathway were focused on limited genes in one or two specific Nicotiana species. With the development of next generation sequencing technology, great progress has been made in getting the tobacco genome. Although seven complete genome sequences in Nicotiana have been released, these are still insufficient for genetic researches of Nicotiana. Compared with whole genome sequencing, RNA sequencing can provide a unique opportunity for genomic exploration in species that do not have a reference genome sequence. It costs less and is more computationally tractable, while can still generate relatively rich information. It has been successfully used in a number of plant species, including Nicotiana, Arabidopsis thaliana, Medicago Sativa and Glycine max.In this study, sixteen Nicotiana species from eight sections were collected and applied to the transcriptome study. I de novo assembled the leaf transcriptomes, and annotated genes according to the NR, Swiss-Prot, KEGG and GO databases. Based on the transcriptome data, I conducted the phylogenetic analysis, correlation analysis between gene expression and nicotine content, WGCNA analysis and important gene analysis. The main conclusions are:(1) A total of 2,167 single copy orthologs were identified and used to construct phylogenetic trees using the Maximum Likelihood method and Neighbor-joining method respectively. The topologies of the two trees were similar. Compared with the previous trees constructed based on morphology or few genes, the trees of the sixteen Nicotiana species constructed based on the transcriptome data were more robust with high bootstrap support, and the phylogenetic relationships of different Nicotiana species were more reliable.(2) N. otophora from section Tomentosae was close to N. tabacum from section Nicotiana. This may be due to the additional genomic contributions from N. otophora during the forming process of N. tobacum, because an introgressed hybrid between N. tomentosiformis and N. otophora was proposed as the paternal parent of N. tobacum.(3) N. bonariensis and N. longiflora having different chromosome number from section Alatae were close. This is different from the previous research results based on the plastid DNA sequences, and further researches are needed.(4) Transcription profile of the sixteen Nicotiana species was obtained, and the correlation between expression of the candidate genes and nicotine content were analyzed. All candidate genes involved in the nicotine biosynthesis showed a positive correlation in general, and genes related to the degradation process displayed a negative correlation overall.(5) A number of genes strongly correlated with nicotine content were identified. They are quinolinic acid phosphoribosyltransferase (QPT2), aspartate oxidase (AO), berberine bridge enzyme-like (BBL) and N-methylputrescine oxidase (MPO). Conversely, putrescine N-methyltransferase (PMT) and isoflavone reductase-like gene (A622) expressed low in all Nicotiana species, indicating that these steps are not active in leaves.(1) Contents of KNOX, antifreeze, N and NBS genes are detected. N. pauciflora and N. undulata had the highest KNOX gene content, N. undulata had the highest antifreeze gene content, and N. bonariensis had the highest N and NBS gene contents.Besides, during the doctoral study period, I analyzed the conservation genetics of two important species based on the plasmon. One is the Procapra przewalskii which is a rare species in China, and listed as endangered in IUCN Red List of Threatened Species, and another is the Apostasia shenzhenica which is discovered in 2011 and named according to the name of the discovery city, and all the wild plants in Orchidaceae are listed in Convention on International Trade in Endangered Species of Wild Fauna and Flora. In the era of molecular biology and genome, plasmon can provide indispensable genetic information for classification, phylogeny and species identification of animals and plants.In this study, the complete mitochondrial genome of Procapra przewalskii was sequenced, and the chloroplast genome of Apostasia shenzhenica przewalskii was utilized. Based on the the mitochondrial genomes of Procapra przewalskii and othe Bovidae species as well as the chloroplast genomes of Apostasia shenzhenica and other Orchidaceae species, the phylogenetic analyses of Bovidae species and Orchidaceae species were condudted. The main conclusions are:(1) Both phylogenetic trees of Bovidae species and Orchidaceae species had high bootstrap support.(2) P. przewalskii and P. gutturosa from Procapra genus were close, while P. gutturosa and Madoqua species were not closest.(3) A. shenzhenica and A. odorata from Apostasioideae subfamily were close, while Apostasioideae and Cypripedioideae subfamilies clustered in different phylogenetic clades.