| Orchids have important economic,cultural,ecological and scientific value.The seeds of orchids are tiny and have no endosperm,and the nutrients stored in the seeds themselves cannot meet the nutritional needs of germination.It is often necessary to rely on the symbiosis of mycorrhizal fungi to provide nutrients for them to complete the seeds germination and the growth and development of protocorms.The symbiosis of mycorrhizal fungi is particularly important for the normal growth and survival of orchids.Our country has rich resources in orchid wild plant.However,little is known about orchid mycorrhizal fungi resources,the symbiotic relationship with orchids,and the significance in orchid biodiversity conservation.More research is needed.In this study,the mature seeds of L.japonica were used as experimental materials,and the fungal strains that could promote the germination of the seeds were screened by tissue culture technology.Using high-throughput transcriptome sequencing technology to obtain the transcriptome information of the seeds of L.japonica and the fungi of Tulasnella at different germination stages under symbiotic and non-symbiotic conditions.Using bioinformatics research methods,the gene expression and gene differential expression of samples at each stage were analyzed in detail,and potential symbiotic genes were initially screened.It is hoped that through the research of this paper,the promoting effect of the fungi of Tulasnella on the germination of L.japonica seeds will be clarified,the symbiotic germination fungal resources of orchid seeds will be enriched,and the regulation mechanism of symbiotic germination of L.japonica seeds will be preliminarily explored at the molecular level.The screened symbiotic-related genes can provide a theoretical basis for the subsequent in-depth elucidation of the nature of the symbiotic relationship between orchids and mycorrhizal fungi and the rational use of orchid mycorrhizal fungi resources.The results of this paper are as follows:(1)One strain(No.65)was selected from the seven Tulasnella fungal strains with the best effect on promoting the germination of L.japonica seeds.The germination rate of L.japonica seeds during symbiotic germination was 85.24%,which was significantly higher than that of other strains.Using this strain,a symbiotic germination system with L.japonica seeds was successfully established.Paraffin section and microscopic observation showed that mycelium could be detected in L.japonica seeds in different germination stages of symbiotic germination,which verified that the symbiotic system was successfully constructed.(2)Taking the symbiotic germination inoculated with No.65 Tulasnella fungus strain as the treatment group and the non-symbiotic germination without Tulasnella fungus as the control group under the same culture conditions,the transcriptome analysis of the two groups of samples from L.japonica seed germination stage 1 was performed.The results show that the number of filtered sequences is more than 45 million,and the mapping rate exceeds 95%.The data quality is good for subsequent analysis.Compared with the control group,most of the differentially expressed genes were mainly up-regulated.The main functions were annotated in biological processes,and the metabolic pathways were mainly starch and sucrose metabolism pathways.Gene regulation in the early stage of L.japonica seed germination is mainly for nutrient supply.11 lectin-related genes,12 nodulin-related genes and 8 GRAS family-related genes were screened,among which TRINITY＿DN43374＿c4＿g3,TRINITY＿DN50768＿c3＿g1 and TRINITY＿DN36162＿c0＿g1 genes were significantly up-regulated.It is speculated that these three genes may be involved in the establishment of the symbiotic germination system of L.japonica seeds and Tulasnella fungi,so they can be focused on in follow-up studies.(3)Since L.japonica seeds did not show any obvious signs of germination under non-symbiotic germination conditions without Tulasnella fungus inoculation,we then conducted a differential comparative analysis of the transcriptomes of samples of L.japonica seeds at four different germination stages under symbiotic germination conditions.The protocorm transcriptome data and the symbiotic Tulasnella fungus transcriptome data at different germination stages were obtained.Among them,the quality of the transcriptome sequencing data of protocorm is good,with more than 44 million filtered sequences,and the mapping rate exceeds 95%,which meets the needs of the subsequent analysis of the experiment;the gene expression of protocorm at different stages varies greatly.Metabolic pathways are mainly enriched in phenylpropane biosynthesis,plant mitogen-activated protein kinase signaling pathway and photosynthesis pathway,and differential gene functions are mainly concentrated in cellular components,indicating that gene regulation at these stages is related to plant morphological development.A total of 20 lectin-related genes,15nodulin-related genes and 12 GRAS family-related genes were screened out.Among them,the expression levels of TRINITY＿DN42119＿c1＿g4,TRINITY＿DN19608＿c0＿g1,TRINITY＿DN37564＿c3＿g6 and TRINITY＿DN30240＿c0＿g1 genes were significantly different at different germination stages,and showed a gradual upward trend in general.Transcriptome data analysis of Tulasnella fungi in symbionts showed that the gene expression changes of Tulasnella fungi in different germination stages were not very different.It is speculated that in the process of orchid seed germination,the role of fungi is to establish a symbiotic relationship with orchid seeds and start the process.Seed germination,once the initiation mechanism is completed,the interaction between the two is relatively stable in the subsequent germination stage,and there is no obvious specific regulation.However,due to the low quality of transcriptome sequencing data for Tulasnella fungi,this conclusion may need to be validated with more exhaustive data. |
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