Bioinformatics analysis of endophytic bacteria related to berberine in the Chinese medicinal plant Coptis teeta Wall

Plant endophytic microorganisms absorb nutrients and prevent pathogen damage, supporting healthy plant growth. However, relationships between endophytic bacteria of the medicinal plant Coptis teeta Wall. and berberine production remain unclear. Herein, we explored the microbial composition of wild-type (WT) and cultivated Coptis teeta Wall. root, stem and leaf, and endophytic bacteria related to berberine. Microbial characteristics of were analyzed by 16S rDNA sequencing, and berberine in roots was analyzed by high-performance liquid chromatography (HPLC). Proteobacteria, Actinobacteria and Bacteroidetes were the major phyla, and Mycobacterium, Salmonella, Nocardioides, Burkholderia-Paraburkholderia and Rhizobium were the dominant genera. Berberine was positively correlated with total P (TP), total N (TN), total K (TK) and available K (AK) in rhizosphere soil, and with Microbacterium and norank_f_7B-8, whereas TK was positively correlated with Microbacterium, TN, AK and Burkholderia-Paraburkholderia. The findings will support further studies on endophytic bacteria and berberine in Coptis teeta Wall., and may promote berberine production.

widely considered to produce the best quality Coptidis rhizome. 48 In addition to use in Chinese medicine, Coptidis rhizome is also a source of  Studies have shown that the root of Coptis teeta Wall. contains 67% berberine, while 55 the stem contains 23% and the leaf contains 11.97%. 56 Coptis teeta Wall. is a shade-tolerant plant that grows at high altitude, in cold 57 mountainous and rainy regions. Coptis teeta Wall. is slow-growing, and it takes 6 to 7 4 58 years after sowing before products can be harvested. The slow growth is often 59 affected by a variety of diseases and pests, such as anthracnose [5]. Its rhizomes are 60 small, and it produces unusual 'breeding branches' that consume a lot of nutrients [6]. 61 Therefore, the yield of Coptis teeta Wall. is very low. Furthermore, due to long-term index, yield, and quality of Coptis teeta Wall. [9], and N, P and K play important 72 roles in vegetative growth, indicating that their combined application may be key to 73 achieving a high yield. Application of fertiliser can significantly increase the yield, 74 and artificial cultivation of Coptis teeta Wall. is the only method capable of meeting 75 demand, hence the need for an improved, standardised approach. 76 In recent years, plant microbiome research has developed rapidly [10,11].

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The present work expands on recent research on the medicinal plant Coptis teeta 86 Wall. by investigating the microbial composition in the root, stem and leaf of 87 wild-type (WT) and cultivated Coptis chinensis. The synthesis of berberine in C. 88 chinensis is likely related to endophytic bacteria, and this is correlated with nutrition. 89 Therefore, in the present study, WT and cultivated Coptis teeta Wall. samples were 90 collected from the country of origin, and the microbial characteristics of root, stem 91 and leaf tissues were analysed by 16S rDNA sequencing. Rhizosphere soil samples of 92 were also collected to measure the content of total P (TP), total N (TN), total K (TK) 93 and available K (AK) in the soil. In addition, the berberine content in roots were 94 analysed by high-performance liquid chromatography (HPLC). immobilised on the chip. Using the DNA fragment as a template, the base sequence 125 immobilised on the chip served as a primer for PCR synthesis, and the target DNA 126 fragment to be detected was synthesised on the chip. After denaturation and 127 annealing, the other end of the DNA fragment on the chip was randomly 128 complementary to another primer in the vicinity, and also fixed to form a 'bridge'.

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PCR amplification produces DNA clusters, and the DNA amplicon was linearised into       OTU level were generated based on the selected distance matrix (Figure 4a). Hcluster 257 can clearly reveal the distances of sample branches, and a hierarchical clustering tree 258 was generated based on the unweighted pair group method with arithmetic mean 259 (UPGMA; Figure 4b). In addition, ANOSIM used the Bray-Curtis algorithm to 260 calculate differences between pairs of samples, and to test whether differences 261 between pairs of groups were significantly greater than intra-group differences (Table   262 1).  (Table 1).  Figure 5d). 295 We analysed the TK, TP, TN and AK content in WT and cultivated rhizosphere  (Table 2).

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Berberine content in roots 303 Next, we analysed the berberine content in the roots of WT and cultivated Coptis 304 teeta Wall. by HPLC, and berberine levels were significantly higher in WT roots 305 ( Figure 6).

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Correlation analysis 307 We used canonical correspondence analysis (CCA) to explore the correlation 308 between endophytes in WT and cultivated Coptis teeta Wall. roots at the genus level,  The closer the R value is to 1, the greater the intra-group differences, and the smaller 552 the R value, the less significant the differences between and within groups.