| Orchidaceae is one of the three major families in the plant kingdom,and most orchids have high ornamental,medicinal and scientific value.At present,some important ornamental orchid plants have achieved tissue culture seedlings,but the tissue culture seedlings grow slowly after transplanting,resulting in high production costs.Under natural conditions,the symbiosis of mycorrhizal fungi is necessary for the germination and protocorm growth of orchids.This interaction is considered to be parasitic orchids on fungi.But is it possible to use mycorrhizal fungi to promote the growth of orchid tissue culture seedlings? Is there a special interaction mechanism between mycorrhizal fungi and orchid seedlings with photosynthetic ability? This paper aims to analyze the stage-dependent molecular reponses in the interaction of Cymbidium hybridum and two different mycorrhizal fungi species.The main results of this paper are as follows:(1)Both mycorrhizal fungi ML01 and Piriformospora indica DSM11827 can form mycorrhizal with Cymbidium seedlings in a confined soil matrix,but the colonization process and growth-promoting effects of this two fungi are different.Piriformospora indica colonized the host roots than that of ML01,but after 7 days of co-cultivation,the colonization rate of ML01 is greater than that of P.indica.Two fungi can promote the absorption of N,P,K,Ca,Mg,Zn in the stems and leaves of Cymbidium hybridum,and promote the absorption of N,P,K,Ca,Zn,Cu in the root system.(2)The whole genome of fungus ML01 was sequenced.After assembly,the genome size of fungus ML01 was 81.39 Mb,51 contigs were obtained,and the length of N50 was3.20 Mb.After gene prediction,a total of 19733 gene encoding proteins were obtained,and the CDS annotation rate was above 90%.Through bioinformatics pipeline,887 secreted proteins and 391 effector proteins were predicted,including 135 PCWDEs,187 apoplast effector proteins,and 69 intracellular effector proteins.(3)The previous Cymbidium root transcriptome were greatly optimized and the nonorchid sequences were removed using the ML01 genome.This transcriptme contained108012 transcripts,with a total sequence length of 143.12 Mb,N50 of 1732 bp,a predicted CDS of 55559,with an average length of 1389.4 bp,BUSCO detection integrity of 85.2%,NR annotation rate of 52.56%,Swiss-Prot annotation rate is 38.34%,GO annotation rate is37.97%.Through amino acid homology analysis(Identity> 50%)and conservative domain prediction,94 Cymbidium sequences highly similar to the known plant symbiotic functional gene cassette sequences were obtained.(4)RNA-seq analysis showed that there were 17,503 differentially expressed genes in orchids treated with ML01 and 12,298 differentially expressed genes in orchids treated with Piriformospora indica.The modules obtained by WGCNA and DEGs at different time points were enriched in GO and KEGG and found to be enriched in the pathways of starch and sucrose metabolism,flavonoid biosynthesis,and phenylpropane biosynthesis;analysis of genes related to homologous orchid symbiosis was found OM symbiosis is related to SLs esterase,ABC transporter,phosphate transporter,ammonium salt transporter,and cytochrome P450 genes.Comparing the molecular responses of the host and two different types of mycorrhizal fungi at different stages of interaction,this study revealed that ML01 has a higher affinity with the host than the endophytic fungus.(5)Analyze the differential genes of the fungi.The different time points of fungal DEGs treated by ML01 were 4787,4481 and 4797,respectively.The different time points of fungi DEGs treated by Piriformospora indica were 3445,3882 and 3991 respectively.After enrichment analysis,it was mainly enriched in cellulose catabolism and fatty acid biosynthesis;the expression patterns of genes related to cellulose degradation were analyzed,and their two different types of fungal invasion processes were different.This study used transcriptome and fungal genome to solve the problem of fungal sequence removal in the process of de novo mycorrhiza assembly,and provided reliable transcripts for studying the symbiotic mechanism of OM mycorrhiza.The OM fungal genome was obtained,which provided a basis for the screening of effector proteins in OM mycorrhizal symbiosis.The changes of some genes and pathways of fungi and orchids in the mycorrhizal symbiosis process were preliminarily analyzed.This study provides us with new insights into the initial understanding of the symbiotic mechanism of OM mycorrhizal and a new method for the rapid growth of seedlings in bottles. |
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