ABSTRACT
Saikosaponin D (SSd) is a naturally active product with strong pharmacological activity found in Bupleurum scorzonerifolium Willd. Studies have shown that endophytic fungi have great potential as sources of natural medicines. Fusarium acuminatum (CHS3), an SSd-producing endophytic fungus, was isolated from B. scorzonerifolium. To elucidate the effect of host plants on the production of SSd in CHS3, CHS3 was co-cultured with suspension cells of B. scorzonerifolium and SSd was detected using high-performance liquid chromatography (HPLC). Transcriptome sequencing (RNA-Seq) of CHS3 before and after co-culture was performed using an Illumina HiSeq 2500 platform. The results indicated that the content of SSd synthesised by CHS3 increased after co-culture with suspension cells of B. scorzonerifolium. Transcriptome analysis of CHS3 with differentially expressed genes (DEGs) showed that 1202 and 1049 genes were upregulated and downregulated, respectively, after co-culture. Thirty genes associated with SSd synthesis and 11 genes related to terpene backbone biosynthesis were annotated to the Kyoto Encyclopaedia of Genes and Genomes (KEGG). Combined with transcriptome data, it was speculated that the mevalonate (MVA) pathway is a possible pathway for SSd synthesis in CHS3, and the expression of key enzyme genes (HMGR, HMGCS, GGPS1, MVK, FDFT1, FNTB) was validated by qRT-PCR. In conclusion, the endophytic fungus CHS3 can form an interactive relationship with its host, thereby promoting SSd biosynthesis and accumulation by upregulating the expression of key enzyme genes in the biosynthesis pathway.
ABSTRACT
Saikosaponin d (SSd) is the main component of Bupleuri Radix, a famous traditional Chinese herbal medicine, with high medicinal value. An endophytic fungus (CHS3) was isolated from Bupleurum scorzonerifolium Willd. in the early stage of our research, and we found that CHS3 could promote the accumulation of SSd in Bupleurum scorzonerifolium Willd. suspension cells (BSS cells). It is of practical significance to identify the mechanism that CHS3 promoted the accumulation of SSd and increased the production of SSd in suspension cells. To search the influence of CHS3 on SSd synthesis in the BSS cells, we co-cultured CHS3 with the BSS cells and compared the SSd content in BSS cells before and after co-culture using high-performance liquid chromatography (HPLC). Then the Illumina HiSeq 2500 was performed to detect the transcriptome of the BSS cells before and after co-culture and analyzed for the KEGG enrichment. The expression of genes involved in SSd synthesis was finally corroborated by qPCR analysis. Among which 11 key genes in connection with SSd synthesis were increased in BSS cells of co-cultured group compared with the BSS cells of the control group. In conclusion, CHS3 could promote the accumulation of SSd in BSS cells, and the molecular mechanism was related to its ability to regulate the MVA pathway, the calcium signaling pathway, and the AMPK signaling pathway by upregulating the expressions of ANT, CypD, CaM, AMPK, AATC, HMGS, HMGR, MVK, MVD, SS, and SE.
Subject(s)
Bupleurum , Drugs, Chinese Herbal , Oleanolic Acid/analogs & derivatives , Saponins , Bupleurum/chemistry , Drugs, Chinese Herbal/chemistry , AMP-Activated Protein Kinases , Molecular Structure , Saponins/chemistry , Gene Expression ProfilingABSTRACT
Saikosaponin d (SSd) is an important bioactive compound of traditional Chinese medicinal plant Bupleurum scorzonerifolium Willd. and exhibits many effects, such as anti-tumor, anti-inflammation and immunomodulatory. Since endophytic fungi possess the natural capacity to produce the similar secondary metabolite to that of their host plants, they are promising as alternative sources of plant bioactive natural products. In this study, in order to search for SSd-producing strains, endophytes were isolated from B. scorzonerifolium and were authenticated by the ITS sequence and the translation elongation factor-1alpha gene (TEF-1α) sequence analysis. The profile of metabolites present in the crude exacts was carried out by ultra performance liquid chromatography time-of-flight mass spectrometry (UPLC/Q-TOF-MS) analysis. The results showed that two strains, CHS2 and CHS3 from B. scorzonerifolium could produce SSd by UPLC/Q-TOF-MS analysis, and the amount of SSd produced by strain CHS2 and CHS3 were about 2.17 and 2.40 µg/mL, respectively. CHS2 and CHS3 showed a close phylogenetic relationship to Fusarium oxysporum and Fusarium acuminatum, respectively. According to our concern, no endophytic fungi capable of producing SSd from B. scorzonerifolium have been found before. Our clear intention was to isolate and identify these endophytic fungi that produce important active secondary metabolites, and then study the strains that produce this compound on a large scale through fermentation or even genetic study, to provide a feasible and more convenient way for the production of SSd.
