Research On Mechanisms Of Symbiosis Formed By Epidendrum Secundum And Tulasnella Spp.

Epidendrum secundum is an epiphytic Orchidaceae species and widely distributed in South America, it blossom all the year round and has an important ornamental value. The tiny seed of orchid is lack endosperm, it could not germinate by itself for undernutrition, the seed and protocorm need mycorrhizal fungi to germinate and develop into seedling. Consequently, the symbiosis is significant to the orchid growth and development. Compared to arbuscular mycorrhiza and ectomycorrhiza, the recognition and symbiosis mechanism of orchid mycorrhiza is relative backward. So, in this study, the plant tissue culture techniques, high-throughput sequencing techniques and bioinformatics were used in this study to establish the symbiotic between mycorrhizal fungi and orchid, obtain the transcriptome information of Epidendrum secundum and Tulasnella calospora in the state of symbiotic and asymbiotic, and analyze the "orchid-mycorrhiza" symbiotic mechanism related genes. In order to understand the recognition and interaction between orchid and mycorrhizal fungi, and molecular mechanisms of symbiosis, and get orchid symbiotic related genes. The main results of this research are follows:(1) Tulasnella spp. co-cultured with Epidendrum secundum, Cymbidium ensifolium, Dendrobium officinale and other orchid seeds and protocorms or rhizomes in three concentrations(0.25 g/L, 2.5 g/L and 5 g/L) oatmeal agar medium. Screened 0.25 g/L as the most suitable medium for co-culturing Epidendrum secundum with Tulasnella spp., and the nutrient concentration in the medium of fungi and plants may decide whether to establish a symbiotic relationship. These six mycorrhizal fungi only symbiosis with seed and protocorm of Epidendrum secundum, and could be identified to species of Tulasnella calospora strains than the strains only be determined on the taxonomic status of the genus were more effective in promoting Epidendrum secundum seed germination or protocorm development, including a Tulasnella calospora(Tco2) the most obvious effect.(2) A total of three groups, each with three copies that nine samples transcriptome sequencing Clean reads of Epidendrum secundum and Tulasnella calospora symbiosis, Epidendrum secundum asymbiosis, and Tulasnella calospora asymbiosis were: 66,691,912, 74,232,026, 60,926,516, 42,809,890, 56,139,672, 45,185,166, 95,270,984, 98,812,276, 112,358,184 item, accounting for the proportion of the raw sequencing data were higher than 94%, and subsequent sequencing analysis to meet quality requirements. Trinity assembled Unigenes 128,115 and 27,873 in Epidendrum secundum and Tulasnella calospora, respectively. In annotation results, the closely related species of Tulasnella calospora is itself, and for Epidendrum secundum is Elaeis guineensis. The gene function of Epidendrum secundum and Tulasnella calospora focused on cellular processes, cellular and binding, and the main metabolic pathways are ribosome and carbon metabolism. The establishment of the symbiotic relationship between Epidendrum secundum and Tulasnella calospora has played a certain role in the level of gene expression.(3) Epidendrum secundum under symbiotic contrast asymbiotic genes expression were based on up-regulation, and that of Tulasnella calospora were mainly on down-regulation. The genes of Epidendrum secundum and Tulasnella calospora after establishing symbiosis significantly increased the expression were related to the catalytic activity. The genes involved in plant-pathogen interactions pathway in Epidendrum secundum symbiotic group were largely induced, and plant took resistance mechanisms to limit the development of fungi, preventing the symbiosis between fungi and plants into saprophytes. Tulasnella calospora symbiotic group involved in carbon metabolism pathway genes were down-regulated expression to inhibit the metabolism of carbon to achieve adaptation to the environment and to improve competitiveness. Understood the Epidendrum secundum symbiotic related genes were PC, calmodulin gene, calcium dependent protein kinase gene and lectin gene. Calcium-dependent protein kinases as plant identification fungal signal transduction, regulation of the early stages of fungus into plant cell colonization, recognized fungi releasing factor and then induced coding early nodulin-like genes expression that related to symbiosis formation and development, whereas during the symbiotic relationship building, lectin gene induced expression to control fungal growth for maintaining symbiotic a stable relationship.(4) Based on the Phalaenopsis equestris genome data, we identified 30 putative PC genes and analysed Phytocyanin gene family in the orchid genome through comprehensive bioinformatics. The PCLD domain of PCs included two highly conserved Cys residues. Nearly half of the PCs are chimeric AGPs, according to its protein backbone they were divided into six groups, Pe PCs protein structure and subfamily classification were highly similar to At PCs. Phylogenetic analysis of At PCs and Pe PCs protein sequences were clustered into seven branches, and indicated that each subfamily was derived from a common ancestor before the divergence of monocot and dicot lineages and that the expansion of the PC subfamilies occurred after the divergence of orchids and Arabidopsis. The family gene structure with two exon one intron type based, relatively conservative. The majority of PC genes were up-regulated expression and the ENODL gene was down-regulated expression in Epidendrum secundum symbiotic, then some PC genes were highly expressed in four tissues but the ENODL genes were low expressed in root tissue that might have infected, suggested that these genes played important roles in orchid mycorrhizal symbiosis formation and we need more research to clear the molecular mechanism of them.