Diversity Analysis Of Endophytes Of Huperzia Serrata And Optimization Of Fermentation Conditions Of Endophytic Bacterium S1026 Producing Huperzine B

Huperzia serrata is a rare medicinal plant in China.Its main active components(huperzine A/B,etc.)have outstanding acetylcholinesterase inhibitory activity,which are important raw materials for clinic drugs for the treatment of Alzheimer's disease.However,the current shortage of wild Huperzia resources,has been unable to meet the growing market demand.The long-term symbiotic relationship between endophyte and host makes some strains produce same or similar bioactive substances as the host.In this study,the diversity and composition characteristics of endophytes in the root,stem and leaf tissues of three-year-old H.serrata were analyzed by high-throughput sequencing technology.The culturable endophytes were isolated from the plant by traditional microbial culture technology,and a bacterial strain capable of producing huperzine B was obtained,and subsequently the preliminary identification and the optimization of fermentation conditions of this strain were carried out.These results laid a foundation for further development and utilization of endophytic bacteria resources in H.serrata.The main results are as follows:(1)The composition and diversity of total endophytic bacteria in the root,stem and leaf tissues of three-year-old H.serrata were analyzed by Illumina Miseq highthroughput sequencing technology.A total of 609,443 effective sequences were obtained,which were divided into 936 OTUs,belonging to 12 phyla,25 classes,83 orders,141 families and 289 genera.At phylum classification level,Proteobacteria was the dominant flora in the root(75.6%),stem(62.2%)and leaf(64.8%)tissues.At genus classification level,Burkholderia-Caballeronia-Paraburkholderia was the most abundant genus in the root tissues,Sphingomonas was the most abundant genus in both the stem and leaf tissues.Ace,Chao1,Simpson and Shannon indexes showed that the diversity and richness of total endophytic bacteria in the leaf tissues were higher than that in the stem tissues,and both were significantly higher than that in the root tissues.Principal coordinates analysis(PCoA)showed that the bacterial communities in the stem and leaf tissues had high similarity,but were significantly different from those in the root tissues.Microbial community function prediction analysis showed that relatively abundant bacterial communities in specific tissue participated in characteristic KEGG pathways,the function of which were similar to that of the specific tissue.For examples,the relative abundance of endophytic bacteria categorized in�energy production and conversion' within the stems and leaves was higher that of the roots,while the roots contained more endophytic bacteria involved in functions such as�carbohydrate and inorganic ion transport'.(2)High-throughput sequencing technology was used to investigate the composition and diversity of total endophytic fungi in the root,stem and leave tissues of three-year-old H.serrata.A total of 692,172 effective sequences were obtained,which were divided into 555 OTUs,belonging to 7 phyla,21 classes,50 orders,94 families and 117 genera.At phylum classification level,Proteobacteria was the dominant flora in the root(75.6%),stem(62.2%)and leaf(64.8%)tissues.At genus classification level,the most abundant genera in the roots,stems and leaves were Oidiodendron,Cladosporium and Phyllosticta,respectively.Alpha diversity analysis showed that the diversity and richness of total endophytic fungi were higher in the leaf tissues than in the stem tissues,and both were a significantly higher than in the root tissues.PCoA showed that the fungal communities in the stem and leaf tissues had higher similarity than those in the root tissues.Spearman correlation analysis among dominant species showed that the relationship among endophytic fungi in H.serrata was complex,but most of them were cooperative(92.1%).(3)In this study,142 strains of endophytic bacteria and 41 strains of endophytic fungi were isolated from H.serrata,and a strain numbered as S1026 producing Hup B was obtained by detecting the fermentation products of each strain.Based on its 16 S rDNA sequence analysis and phylogenetic tree analysis,the S1026 strain was identified as Kluyvera.(4)Furthermore,the fermentation medium components and culture conditions of S1026 strain were optimized.The results of orthogonal test showed that when carbon source was 7 g/L sucrose,inorganic salt was 7 g/L potassium chloride and nitrogen source was 5 g/L soybean powder,the yield of Hup B was the highest,which was 18.93?g/L,which was 1.98 times of the control group(9.57 ?g/L).The optimum culture conditions were determined by single factor experiments,and the results showed that when the initial p H value of the fermentation medium was 8.0,the culture temperature was 37?,and the shaking speed was 150 r/min,the Hup B yield of endophytic bacterium S1026 was the highest,which was 21.13 ?g/L,about 2.21 times that of the control group.