乌头内生细菌群落结构及其与宿主生物碱合成关系研究

【作者】 王娜；

【导师】 崔浪军；

【作者基本信息】 陕西师范大学 ， 生物化学与分子生物学， 2014， 硕士

【摘要】 内生细菌广泛存在于陆地、水生健康植物体内,与宿主形成了相互依存、协同进化的关系。大量的研究表明,内生细菌具有促进宿主生长、提高宿主抵御生物胁迫与非生物胁迫的能力,进而提高宿主环境适应性。药用植物有效成分不仅广泛用于医药、食品、化妆品等领域,而且在宿主次生生长阶段与环境相适应的过程中起着关键作用。因此,阐明药用植物内生细菌与宿主有效成分的关系是揭示药用植物宿主与内生细菌互作机制的关键所在。附子是毛茛科植物乌头(Aconitum carmichaeli Debx.)子根的加工品,是我国传统名贵大宗药材,被历版《中国药典》收录。附子的有效成分主要为新乌头碱、次乌头碱和乌头碱等生物碱类,具有回阳救逆,补火助阳,散寒止痛等功效,用于亡阳虚脱,肢冷脉微等疾病的治疗。乌头是我国乌头属中分布最广泛的种,据《中国植物志》记载,在我国不同区域,出现了黄山乌头(A. carmichaeli var. hwangshanicum)、展毛乌头(A. carmichaeli var. truppelianum)、毛叶乌头(A.carmichaeli var. pubescens)等变种,这些乌头变种的根系都可加工成附子。另外,大量的研究表明,乌头中的各类生物碱对人体、动物和微生物均有很高的“毒性”,与此同时,乌头体内依然生活着大量的内生细菌。那么均能入药的乌头及其变种内生细菌群落结构之间的差异是什么?是否存在与生物碱合成密切相关的内生细菌?本文以采自陕西汉中、陕西户县、安徽黄山、浙江临安、甘肃兰州的乌头(即草乌)、黄山乌头、展毛乌头和毛叶乌头为材料,在研究分析其内生细菌群落异同的基础上,进一步“寻找”与生物碱合成密切相关的内生细菌,试图为乌头生物碱的积累与其内生细菌之间关系的揭示提供一定依据。本文的主要研究内容和结果如下：1.高效液相色谱法测定乌头及其变种根、茎、叶总生物碱的含量,结果表明乌头及其变种全株总生物碱含量最高是黄山乌头,总生物碱含量最低的是毛叶乌头。乌头及其变种不同组织部位总生物碱含量差异较大,根中总生物碱含量较茎和叶最高。2.以表面消毒完善的乌头根、茎、叶为材料,提取各个样品的DNA,针对各个样品变异较大的V4区(515F-907R),设计引物,进行16S rDNA-PCR扩增,随后进行高通量测序。分析研究乌头及其变种内生细菌多样性：(1)乌头及其变种的内生细菌类群由4门,9纲,16目,22科,36属组成；优势菌门是变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes);最优势属为mitochondria_norank(变形纲门)。(2)在整株水平上,黄山乌头内生细菌多样性最丰富,其次是展毛乌头,最低是汉中产草乌。菌种分布均匀度最高是汉中产乌头,其次是展毛乌头,最低是黄山乌头。(3)PCA结果显示乌头及其变种内生细菌属聚为一类,说明乌头及其变种内生细菌属种类相似度非常高。(4)乌头不同组织部位中根中内生细菌物种丰富度高于茎和叶,叶中菌种分布均匀度高于根和茎。3.在整株水平上,乌头及其变种内生细菌共同属有17个,生丝薇属和Massilia,二者与乌头总生物碱含量呈显著正相关。不同组织部位水平,生丝微菌属与乌头根中总生物碱呈显著正相关；黄色单胞菌属与茎中总生物碱相关的内生细菌属呈显著正相关,mitochondria_norank与茎中总生物碱相关的内生细菌属呈极显著负相关,WCHB1-60_norank与茎中总生物碱相关的内生细菌属呈显著负相关；假单胞菌属与叶中总生物碱相关的内生细菌属呈显著正相关。本文的创新之处在于：(1)全面准确描述乌头及其变种的内生细菌群落多样性及类群组成；(2)筛选出与乌头主要有效成分生物碱合成密切相关的内生细菌类群。更多还原

【Abstract】 Endophytic bacteria widely exist in terrestrial, aquatic healthy plants. It is an interdependent and Co-evolution relationship between endophytic bacteria and the host. Multiple studies found endophytic bacteria can promote the growth of host, enhance the ability of anti-biotic and abiotic stress, improve the environment adaptability of the host. The effective ingredients in medicinal plants are not only widely used in the fields of medicine, food cosmetics, but also play a key role in the adapting environment in the process of the host secondary growth stage. Therefore, to clarify the relationship between endophytic bacteria and the host effective ingredients is the key to reveal the interaction mechanism of medicinal plants and endophytic bacteria.Aconite, the traditional rare medicinal material in China, is the processed products of A.carmichaeli Debxsub. root which is also included by calendar version "China Pharmacopoeia". The effective components of aconite mainly are mesaconitine, hypaconitine aconitine alkaloids and etc. Aconitum is one of the most widely distributed species of aconitum in China. According to the "China flora", A. carmichaeli var. hwangshanicum, A. carmichaeli van truppelianum, A. carmichaeli var. pubescens and etc appear in different regions of China. The roots of aconitum variants can be processed to be aconite. In addition, a large number of studies show that all area of aconitum alkaloids has very high "toxicity" in human, animal and microorganism. However, a lot of endophytic bacteria still live in aconitine. What is the difference of endophytic bacterial community structure of aconitum and aconitum variants? Whether it has a close relationship between endophytic bacteria and alkaloid synthesis or not?In this study, Aconitum, A. carmichaeli var. hwangshanicum, A. carmichaeli var. truppelianum, A.carmichaeli var. pubescens, collected in Hanzhong Shaanxi, Huxian Shaanxi, Ling’an Zhejiang, Lanzhou Gansu. By analyzing the similarities and differences of the endophytic bacterial communities, we further find the endophytic Bacteria closely related to alkaloid synthesis. The results establish the basis for revealing the relationship of aconitum alkaloids accumulation and endophytic bacteria. The main research contents and results are as follows:1. We determine the contents of total alkaloids in roots, stems, leaves of aconitum plants by HPLC method. The content of total alkaloids of A. carmichaeli var. hwangshanicum is the highest in the aconitum and variants, and A.carmichaeli var. pubescens is the lowest. Moreover, the content of total alkaloids is quite different in different tissues, the higher is in the root than the stems and leaves.2. With the surface disinfection, aconite root, stem, leaf were used as material of this study. After extracting the DNA of various samples, we designed a pair of primers for large variability V4region (515F-907R) of each sample, performed16S rDNA-PCR amplification, followed by high-throughput sequencing. Using high-throughput sequencing technology analysis bacterial diversity in the aconitum and variants:(1) The endophytic bacteria community structure in the aconitum and variants is4phylums,9classes,16orders,22families,36genuses. The dominant bacteria phylums are Proteobacteria and Bacteroidetes. The most dominant genus is mitochondria_norank.(2) The diversity of endophytic bacteria in A. carmichaeli var. hwangshanicum is the highest, followed by A. carmichaeli var. truppelianum and aconitum in Hanzhong is the lowest. The species distribution evenness of aconitum in Hanzhong is the highest, followed by A. carmichaeli var. truppelianum. That of A. carmichaeli var. hwangshanicum is the lowest.(3) The PC A analysis showed that there were remarkably similarities of endophytic bacteria in the aconitum and variants.(4) The endophytic bacteria diversity in the root is greater than stem and leave of the aconitum and variants. Leaf species distribution uniformity is higher than that of root and stem.3. There are17kinds of endophytic bacteria of the aconitum and variants in common. The correlation analysis results showed that there was significant positive correlation with the total alkaloids content of the aconitum and Hyphomicrobium, Massilia. The endophytic bacteria common genus of the aconitum and variants in different tissues and the total alkaloids distribution correlation analysis results showed: Endophytic bacteria of the aconitum and variants have6common genuses in root. The bacterial genus correlating with the total alkaloids in root is Hyphomicrobium, and it shows significantly positive correlation. Endophytic bacteria of the aconitum and variants in stems have6common genuses, there was significant positive correlation with the total alkaloids content of the aconitum and Hyphomicrobium, there was significant negative correlation with the total alkaloids content of the aconitum and WCHB1-60_norank, there was significant very negative correlation with the total alkaloids content of the aconitum and mitochondria norank. Endophytic bacteria of the aconitum and variants in stems have3common leaves, there was significant positive correlation with the total alkaloids content of the aconitum and Pseudomonas.Innovation of the research:(1) This paper fully and accurately described the diversity and community structure of the endophytic bacteria from aconitum and its variants.(2) We screened out the endophytic bacteria is closely related with the progress of aconitum alkaloid synthesis which is the main active components.更多还原