Study On The Structure Of Endophytic Bacteria Community Of Aconitum And Its Relationship With Host Alkaloid Synthesis

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.