Research On Symbiotic Relationship Of Dendrobium Sp.and Tulasnella Calospora

Dendrobium sp.,one of the most important genera in the family,is mainly distributed in tropical and subtropical Asia.In China,Dendrobium is mainly distributed in south western provinces and Taiwan island.Orchids have unique modes of acquiring nutrients(mycoheterotrophy)such that seed germination depends on essential nutrients supplied from mycorrhizal fungi.As the seeds of orchids are minute and contain limited energy reserves,their colonization by a compatible fungus is essential for germination under natural conditions.So far,various mycorrhizal fungi have isolated from orchids,but the molecular events involved in the interaction between fungus and plant during this process are poorly understood.In this present study,a symbiotic cultivation system of Dendrobium and mycorrhiza fungi was established.To investigate the expression profiles of the plant and fungi under symbiosis condition or non-symbiosis condition,then isolate the genes involved in symbiotic germination,RNA-seq method was applied to construct libraries of Dendrobium seed and mycorrhiza fungi in different conditions.Based on the RNA-seq data,bioinformatics methods were used to predict genes involved in symbiotic germination at genomic level and analyze molecular regulatory mechanism of Dendrobium and mycorrhiz asymbiotic system in depth.The main research results are as follows:1.Isolated 40 endophytic bacteria form 6 samples of Cymbidium goeringii and C.faberi,and these endophytic bacteria could divid into 15 different genera.3 potential new endophytic bacteria were also found in this study.2.A Tulasnella calospora isolated from C.goeringii in Suizhou,Hubei province,was used to establish the symbiotic germination with Dendrobium seeds.The seeds were confirmed to have a symbiotic relationship with fungi by using trypan bluedye exclusion.After the procedures of amplifing the ITS region,sequencing PCR product,and sequence alignment,the fungus in seeds was verified to be T.calospora3.Nine samples were divided into three different groups based on different processed ways: seeds under symbiotic germination codition(DoTcg),seeds under asymbiotic germination codition(Do),fungi under asymbiotic germination codition(Tcg).Each group contained 3 biologica reduplicates,and a total of 96 G high quality transcriptome sequencing data was obtained.The values of repeat coefficient of biological samples(R2)were ahout 0.97-0.99,indicating a good reproducibility.The analysis of Dendrobium transcriptome was based on reference genome,about 45%-60% reads were mapped to Dendrobium genome,70% of reads were located in exon,and the intron had the lowest reads numer.About 25% of reads were located in intergenic region.Based upon the newly pulished Dendrobium gene annotation file,new functional genes were found in this study,and 16 872 known genes were got structure optimization.4.Transcriptome sequencing data of Tcg were assembled de novo assembly strategy,a total of 58967 transcripts sequences were obtained.Among these transcripts sequences,the number of unigenes with length greater than 1 000 bp was 15 257,accounted for 25.87%.While the length of N50 unigene was 1 488 bp.The number of SNP site in the 6 Tcg transcriptome sequencing is 65 000 to 70 000,about 15 000 to 17 000 SNP site were part of non-coding sequences and 50 000 were belong to coding sequences,most of the SNP sites were synonymous substitutions.5.Between the expression profiles of symbiotic samples and non-symbiotic samples,five categories of differentially expressed genes(DEGs)related to hormone signal transduction pathways were predicted in DoTcg group.Among these genes,9 genes were related to ABA signal transduction pathway,4 were related to GA signal transduction pathway,15 were related to IAA signal transduction pathway,one was related to brassinosteroid signaling pathway and 10 were related tocytokin in signaling pathway.6 DEGs related to glycine/serine hydroxyl methyl transferase were shown significant difference in Tcg group.And 4 gibberellic acid receptor protein genes,7 genes belong to AUX/IAA family,3 genes coding auxin response and receptor proteins were also shown significant difference.In DoTcg group,some genes related to mineral elements transhipment and assimilation were both found in seeds and T.calospora.In the 6 Dendrobium transcriptome data libraries,a total of 333 transcription factors spread over 44 categories were found differential expressed between seeds under symbiotic germination codition and seeds under asymbiotic germination codition.These genes may envolveing in establish symbiotic germination system.6.Bioinformatics methods were used to search the Phytocyanin gene members of DoPCs and DoWRKYs in the database of Dendrobium whole genome,and then the conserved domain,phylogenetic profiling and structural characteristics of the two familes was predicted.Finally,38 DoPCs were expected to have conserved PCLD domains and two Cys residues,belonging to four subfamilies.Among these DoPC proteins,19 DoPCs contained four copper ligands(His?Cys?His?Gln/Met),32 DoPCs were predicted to have N-terminal signal peptides,22 DoPCs had putative C-terminal glycosylphosphatidylinositol-anchor signals,and 27 DoPCs had putative arabinogalactan glycomodules.52 WRKY family genes of Dendrobium were surveyed for the first time.Conserved domain,phylogenetic,exon–intron construction,and expression analyses were performed for the DoWRKY genes.Two major types of intron splicing(PR and VQR introns)were found,and the intron insertion position was observed to be relatively conserved in the conserved DoWRKY domains.