Construction Of Ficus Genome Database And Diversity Analysis Of Terpenoid Synthase Gene Famil

The genus Ficus L.belongs to the family Moraceae,including arbors,shrubs,and vines,and plays an important role in tropical and subtropical forest ecosystems.Because of the unique syconium and special pollination pattern,the mutualistic symbiosis system between Ficus plants and their pollinating insects,fig wasps,has become a classic model for studying the co-evolution of plants and insects.As a highly specific symbiotic system,the molecular driving mechanism behind the fig-fig wasp symbiosis has been an unsolved problem in the fields of ecology,reproduction and evolutionary biology.Terpenoids,components of volatile organic compounds,have long been considered to play an important role in attracting insects for pollination.To study the evolutionary significance of these compounds in the fig-fig wasp obligate symbiosis system,we used the bract samples of 24 Ficus species collected in South China and Southeast Asia to perform transcriptome sequencing.Based on the published genomes and related information of five species of Ficus,we built a comprehensive genomewide database of Ficus plants and an analysis platform for the terpenoid gene family.At the same time,using the in-depth mining of transcriptome data,we also comprehensively compared the phylogenetic relationships and genetic diversity characteristics of the terpenoid-encoding genes in different subgenera and subgroup species of Ficus.The main results are as follows:1.We constructed a comprehensive Ficus Genome Database(FGD,http://www.ficusgd.com).The database is built based on the CentOS 7.8 system using the tripal 3 toolkit,and the implementation of the relevant functional modules is based on Perl/CGI technology,while html+css+javascript is used for front-end web presentation.FGD contains whole genome data of five Ficus species and RNA-seq data of fig bracts of 24 Ficus species,with 174,176 mRNA,171,752 CDS and 702,402 Unigenes sequences.Meanwhile,to facilitate community communication,FGD embeds 11 bioinformatics tools or programs(Blast,JBrowse,GBrowse,Tripal Gene Expression,Syntenic View,GO enrichment,Pathway enrichment,TPS Family analysis,Biochemical pathway)that can provide several operations such as gene annotation,expression and transcriptome profile display,tRNA and SSR retrieval,and 183,625 homologous pairs match among five Ficus species.Here,three modules for GO enrichment,Pathway enrichment and TPS family analysis are personalized platformbased developments.2.The raw reads of 24 transcriptomes of fig bracts at female phase were de nono assembled,and 248 terpene synthase(FTPS)genes were identified from the transcriptomes.Comprehensive analysis of the sequence characteristics,phylogenetic relationships,gene expression patterns and evolutionary selection differences of these 248 genes revealed that the FTPS genes are mainly class I synthase genes,which specifically encodes sesquiterpenes,monoterpenes,and diterpenes.The 248 FTPS genes could be classified into 33 orthologous genes with different expression levels and profiles among 24 Ficus plants.In addition,except for a few genes with positive selection sites or slightly faster evolutionary rates,most of these FTPS genes have undergone purifying selection.These studies suggest that the complex combination and differential expression of multiple FTPS gene may be an important factor contributing to the diversity and specificity of terpenoids in different Ficus species.3.The gene expression level and protein product of FTPS were verified by qRTPCR and Escherichia coli prokaryotic protein expression.Quantitative detection of the expression of eighteen FTPS genes in fig bract samples of three species(F.hirta,F.triloba,and F.hispida)showed that the expression patterns were highly consistent with the results of transcriptome analysis.At the same time,a predicted linalool synthase gene(GenBank No:OP204328.1)in F.microcarpa was used for protein expression and purification in vitro,through incubation with the substrate Geranyl diphosphate and a series of experiments to obtain purified linalool.The results further verified our bioinformatics analysis at the protein product level.In summary,this study not only creates a new database platform for sharing comprehensive data on genome,transcriptome and related information of the Ficus genus,but also deepens the understanding of the diversity and gene characteristics of Ficus TPS gene family,and provides a theoretical basis for elucidating the evolutionary characteristics and maintenance mechanisms of the fig-fig wasp symbiotic system.