Endophytic Bacterial Diversity And Cloning?Bioinformatics Analysis Of Squalene Epoxidase(SE) Gene In Panax Japonicus C.A.Meyer

Panax japonicus C.A.Meyer is a rare species of traditional Chinese medicinal material in China,also know as�king of herbs',It belongs to perennial herbal medicinal plant of the genus Panax of family Araliaceae,which is widely distributed in China,North Korea and Japan.The Southwestern of China is the main biodiversity distribution centers of Panax japonicus.Panax japonicus is rich in triterpenoid saponins,which is the primary medicinal active substance,with various clinical pharmacological effects such as promoting the secretion of body fluid and stopping bleeding,invigorates the blood to dispel the stasis,and nourishing and build up the body.Saponin contents of the rhizome part accounts for 15%of the total saponin content of Panax japonicus,which is 2-7 times that of panax ginseng and 3 times as much as that of Panax quinquefolius.It is endangered by people's extensive excavation and consumption due to its high pharmacological and medicinal value,and the wild Panax japonicus have a long growth period,which cannot meet human needs for their medicinal properties.In recent years,there have been many researches on the Germplasm resources,Medicinal chemical ingredient and Pharmacological activities of Panax japonicus,but there have been no reports on its endophytic bacterial diversity and community structure and cloned of Squalene epoxidase gene.Therefore,Panax japonicus was used as the experimental material to study the endophytic bacterial diversity and community structure of Panax japonicus.The SE candidate gene of Panax japonicus was cloned and its sequence was analyzed by bioinformatics.Through research,the following research results were obtained:1.Take fresh roots,stems,leaves,fruits,and rhizomes of Panax japonicus as materials,five samples were generanted for total genomic DNA extraction as templates,16S rDNA V3-V4 region PCR amplified the genomic DNA of Panax japonicus.1401699 Effective tags were obtained after sequencing,and the mean length of effective tags reads was 405bp.A total of 567 OTUs were obtained from 25 samples,including 63 OUTs are annotated to 8 phylums,which are Proteobacteria,Bacteroidetes,Firmicutes,Actinobacteria,Deinococcus-Thermus,Chloroflexi,Verrucomicrobia and Fusobacteria.2.There were variances of endophytic bacteria community structure in all groups of Panax japonicus.The community structure of the endophytic bacteria in the fruits is significantly variation from that of the rest which maximun richness;the dominant bacteria in the rhizomes are concentrated in the Proteobacteria,and the flora is significantly different from the rest that minimun richness;and the bacteria communities composition of the roots,stems and leaves is relatively similar,mainly concentrated in Proteobacteria and Bacteroidetes.The distribution of different bacteria groups in the five parts of Panax japonicus has certain differences.The Proteobacteria are mainly distributed in the roots,stems,leaves and rhizomes;Firmicutes are mainly distributed in stems and fruits;Bacteroidetes are mainly distributed in fruits and rhizomes;Deinococcus-Thermus are mainly distributed in stems,leaves,and rhizomes,not found in the fruit;Actinobacteria are mainly distributed in rhizomes,not distributed in leaves and fruits.Chloroflexi and Fusobacteria are found only in rhizomes and stems,respectively.Its microbial diversity and richness are expressed as fruits>stems>roots>rhizomes>leafs.3.The Panax japonicus SE candidate gene was cloned according to the reported Panax notoginseng squalene epoxidase gene-specific primer through NCBI.The results showed that the full-length SE gene of the Panax japonicus was 1632 bp,was named PjSE.PjSE contains 6 bp5'-UTR,6 bp 3'-URT,and 1620 bp ORF,the Number of amino acids is539,including the start codon ATG and the stop codon TAA.The Molecular weight of the SE protein is 59380.81kD,the Theoretical pI is8.72,the Formula is C₂₆₇₉H₄₂₃₂N₇₁₈O₇₅₉S₂₃,and the Total number of atoms is 8411.The Grand average of hydropathicity of Pj SE is 0.040,Among the amino acid residues,A50?E?is the most hydrophilic?MIN:-3.556?and A39?F?is the most hydrophobic?MAX:3.0?,and the number of hydrophobic amino acids is more than hydrophilic amino acids.Pj SE protein Instability index is 42.63 and the Aliphatic index is 97.16.4.Bioinformatics analysis indicated that the amino acid sequence of PjSE candidate gene was 99%,98%and 98%homologous to Panax ginseng,Panax notoginseng and Panax vietnamensis,respectively.PjSE has a conserved domain FAD binding site,there are 4 transmembrane domains and 7 motif sites.PjSE protein has random coil and alpha helix as the main constructional elements,and extended strand and beta turn are dispersed throughout the protein that accounting for 40.82%,37.48%,15.77%and 5.94%,respectively.the protein can folding properties to form the typical three-dimensional SE structure.