| Fungi polysaccharides is an important kind of bioactive polysaccharides, which is isolated and purificated from sporophore, mycelium or fermentation liquid of fungi. It has functions in promoting immune, anti-tumor, anti-virus, anti coagulation, hypogly-cemic, hypolipidemic, controlling cell division and differentiation, delay aging, etc. Oligosaccharides promoted polysaccharides applied in plant protect. It has been known that oligosaccharides can induce plant resistance, known as sugar chain plant vaccine. Study on inhibiting plant virus activity of fungi polysaccharides is less in addition to Lentinus edodes polysaccharides. Plant virus is difficult to control because of its absolute internal parasitism and systematic infection, which can been prevented and controlled using immune activity of fungi polysaccharides. But the research mainly focused on defensive enzymes and effect, the molecular mechanism of fungi polysaccharides inhibiting plant disease is less. The thesis chose Coriolus versicolor polysaccharides for pharmaceutical object(1000 ?), selected Tobacco mosaic virus(TMV, the most representative plant viruses) as control object, set 5 treatments: sparying water( as control), spraying C. versicolor polysaccharides, spraying C. versicolor polysaccharides before inoculating TMV 24 hours, inoculating TMV, spraying C. versicolor polysaccharides after inoculating TMV 24 h, repeated 3 times, mixed samples to extract RNA after treatment 72 h. Transcriptome and small RNA sequencing were used to study inhibiting TMV molecular mechanism of C. versicolor polysaccharides and their interaction(agents, plant, and virus), which is expected to explain C. Versicolor polysaccharides inhibiting plant virus. The main results of the thesis are as follows:1. Through molecular identification of fungal rDNA-ITS, ITS region length of Qinling strain IS385 is 740 bp, which homology identification reach to 99% blasted to 12 kinds of C. versicolor strains in NCBI, and GC content is 47.9%. IS385 was identified as C. versicolor Qinling strain, which enriched germplasm resources of macrofung.2. Through transcriptome sequencing, clean data of the samples are up to 20.56 Gb, the percentage of Q30 bases are more than 88.54%, GC content are 44.04%. The clean reads mapped ratio to Nicotiana benthamiana reference genome are from 75.68% to 85.92%. The uniq mapped ratio to N. benthamiana reference genome are from 71.21% to 79.18%. Single nucleotide polymorphism(SNPs) were found more than 40,000, mostly located in the gene. Transformation(62.5%) is more than transversion(37.5%), and heterozygous type accounts for about 58%. Novel predict alternative splicing events have about 31,000, of which spraying C. versicolor polysaccharides(T02) and spraying C. versicolor polysaccharides before inoculating TMV 24 hours(T03) have no significant difference, but is 845 more than sparying water(T01); and inoculating TMV(T04) and spraying C. versicolor polysaccharides after inoculating TMV 24h(T05) have no significant difference, but is 3128 more than sparying water. Up to 22783 genes were optimized in N. benthamiana. 2837 novel genes were found, 1522 of them were annotated.3. Ten different expression genes set were obtained by transcript quantitative analysis. Compared with spraying water(T01), the different expression genes of spraying C. versicolor polysaccharides after inoculating TMV 24h(T05), spraying C. versicolor polysaccharides before inoculating TMV 24 hours(T03), inoculating TMV(T04), and spraying C. versicolor polysaccharides(T02) have 5032, 3124, 1549, and 341. It showed that C. versicolor polysaccharides will mobilize lots of gene transcription and obviously immune regulation effect once virus stress occur. Through functional annotation and enrichment of different expression genes, C. versicolor polysaccharides has obvious immune regulation activity mainly involving ribozyme activity, photosynthesis, energy metabolism,and virus induced gene silencing in host and virus interactions, inducing ascorbate and aldarate metabolism, phenylpropanoid biosynthesis, stilbenoid, diarylheptanoid and gingerol biosynthesis, plant-pathogen interaction, plant hormone signal transduction, virus induced gene silencing, and production of miRNAs involved in gene silencing by miRNA, etc. Proteins interaction network of different expression genes can predict genes regulation pathway.4. Through small RNA sequencing, the clean data of spraying water( as control)(S01), spraying C. versicolor polysaccharides(S02), and inoculating TMV(S03) are greater than 15.44 M, Q30 bases are more than 93%. The public and unique sRNA sequences between samples were to gather statistics. 898 kinds of miRNA were detected and analyzed by MiRDeep2 software, among which 164 were known mi RNA, 654 novel miRNA were predicted. The mi RNA base preference, base editing, and family analysis were analyzed.5. Through miRNA quantitative analysis, the different expression mi RNA of spraying water(S01) and spraying C. versicolor polysaccharides(S02), spraying water(S01) and inoculating TMV(S03), spraying C. versicolor polysaccharides(S02) and inoculating TMV(S03) were screened out 41, 60 and 74 species. Using TargetFinder software, target genes of the known miRNA(104/164) and new prediction miRNA(227/654) were predicted 263 and 666, a total of 913 target genes, of which 871 target genes were annotated function. Through the candidate target genes enrichment of different expression mi RNA, C. versicolor polysaccharides has immune regulation function mainly involving enzyme activity, prosthetic group, amino acid and nucleic acid metabolism, stress response, and molecular function regulation etc.6. Through transcriptome and small RNA association analysis, the different expression up and down genes species combinated with different expression miRNAs between spraying water(S01) and spraying C. versicolor polysaccharides(S02), spraying water(S01) and inoculating TMV(S03), spraying C. versicolor polysaccharides(S02) and inoculating TMV(S03) are up to 9, 21, 18. In the same way,the different expression up and down mi RNA species combinated with different expression genes between samples are up to 29, 25, 38. The known three modes of miRNA regulation modes possibly exist in N. benthamiana. MiRNA-mRNA control network were generated and visual editing using Cytoscape software. Members of nta-mi R172 and nta-miR156 family are predicted as the key miRNAs in gene expression regulation.7. Through sRNA deep sequencing technology, sRNA(not matched to host N. benthamiana genome) were alligned to GenBank virus RefSeq nucleic acid database(allows one mismatch) using Bowtie software. Viral siRNA of samples possible infected by virus were preliminary identificated, which were spliced into 1650 contigs using Velvet splicing software. The 1650 contigs were annotated, classified and proved possible detected 18 kinds of viral sequences, of which 13 kinds of viruses have more than 5 contigs, including TMV accounted for 66.55%(1098/1650). The contigs alligned to other kinds of virus need to verify. The mechanism and cause of its formation are worth attention.The study proved that C. versicolor polysaccharides can induce N. benthamiana resistance genes to express inhibiting TMV infection by means of complex molecular regulation pathway, which enriched fungi polysaccharides inhibiting plant virus theory, and provided the basis for research and development of fungi polysaccharides. |
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