Improvement Of Salt Tolerance Mechanism Of Wild Barley By Epichlo? Endophytic Fungi Based On MRNA And MiRNAomics

In this project,our aim is to eucidate the underlying mechanism in the improved salt tolerance of wild barley-endophyte synbiont at transcriptomic or small RNA level.The characterization of the mechanism was studied by the combined approach of high-throughput sequencing of mRNA and miRAN and some physiological attributes.The main results are as follows:1.In the greenhouse,homologous wild barley?E+?and non-bacterial?E-?plants were planted in half hoagland nutrient solution.After microscopic examination confirmed the endophytic fungi,the wild barley was treated with 200mmol/L sodium chloride for 6 days,and the stems and leaves were taken and quickly frozen in liquid nitrogen and stored in a-80 degree refrigerator.The physicochemical indexes were determined by using the leaves.The results showed that the endophyte increased the content of chlorophyll a,b and carotenoids contents under 200 mmol/L salt stress.The activities of antioxidant enzymes SOD,CAT and POD in the wild barley were higher in the E+ plants than E-plants.And endophytic fungi increased the contents of some osmotic adjustment substanc such as proline and soluble sugars and reduced water loss in leaves.2.The four samples of wild barley was carried out by high-throughput sequencing,and the mechanism of the effects of salt and endophyte on differentially expressed genes in wild barley plants was determined.With the barley genome as the reference genome.The gene expression of wild barley plants was analyzed by bioinformatics.The number of differentially expressed genes was more up-regulated expression in E+ wild barley than in E-wild barley;the effect of salt stress was that the down-regulated expression gene was more than the up-regulated expression.The total number of differentially expressed genes associated with endophyte was 598 and associated with salt was 401.Functional analysis of differential genes revealed that the most abundant pathway for differential gene expression was the metabolic pathway.Endophytic fungi also significantly affected plant immunity,plant-pathogen interaction,fungi,plant hormone signal transduction,starch and sucrose metabolism and other pathways.Comprehensive analysis of differential genes related to jasmonic acid synthesis,suggesting that endophytic fungi affect the synthesis of host jasmonic acid by regulating the metabolic network of jasmonic acid-related pathways.3.Symbiosis were found to significantly affect fungal gene expression by sequencing high-throughput transcriptomes of in vitro endophytic fungi and comparing them with symbiotic endophytic fungi.Compared with vitro endophytic fungi,symbiotic fungi were genes whose expression was up-regulated less than that of down-regulated expression.E+CK vs Pure Fungi had 1532 total difference endophytic fungi genes,E+200 vs Pure Fungi had 1463 total differential genes.Functional clustering of differentially expressed endophytic fungi genes showed that symbiotic effects with the host significantly affected glucose metabolism,lipid metabolism,ribosomes,secondary metabolites,purine and pyrimidine metabolism,amino acid metabolism,signal transduction,oxidative phosphorylation,vitamins,Plastid,peroxisome,etc.pathway.CAT and SOD related genes were up-regulated in symbiotic fungi compared with in vitro fungi.In the analysis of the differential gene function of the plant wild barley,no significant up-regulation of CAT and SOD-related genes was detected,but in the results of CAT and SOD enzyme activities E+ were higher than E-in the leaves of plants under salt stress.The results of this study were very consistent.The molecular mechanism of endophytic fungi promoting the increase of host CAT and SOD activity under salt stress was analyzed from the gene level,mainly derived from the up-regulated expression of endophytic fungi-related genes.4.Through high-throughput small RNA sequencing of four sample wild barley plants,the post-transcriptional regulation mechanism of small RNA on salt tolerance of wild barley plants was determined under the influence of salt and endophytic fungi.The obtained effective sequences were aligned to the barley genome and Rfam database.A total of 257 new mature miRNAs were predicted,and 316 known and new miRNAs were identified.Target prediction of differentially expressed miRNAs was done in this study and performed GO and KEGG functional enrichment on target genes.This study detected known differentially expressed miRNAs associated with salt resistance,including hvu-miR156 and hvu-miR6209.The hvu-miR5050,hvu-miR5049 d,hvu-miR6191,hvu-miR6199,hvu-miR6214 were detected in association with endophytic fungi.This study found that there were 7 differentially expressed novel miRNAs associated with salt and 15 novel miRNAs associated with endophytic fungi.The differentially expressed miRNAs was performed GO and KEGG functional analysis on the target genes.It was found that both salt and endophytic fungi lead to small RNAs to regulate target genes in metabolic pathways,biosynthesis of secondary metabolites,plant hormone signaling,RNA transport,purine metabolism,endoplasmic reticulum protein processing,and splice.In addition,small RNA regulated some amino acid,carbohydrate,thiamine,and flavonoid-related target genes under salt stress.Small RNA regulated target genes in amino acids,pyrimidines,ketones,phenylpropanoids,N-polysaccharides,methyl butyrate under the influence of endophyte.And the endophyte also affected photosynthesis,oxidative phosphoric acid,RNA degradation,ribosome,ubiquitin-mediated protein degradation,plant pathogen interactions,and peroxisomal target genes.The mechanism of miRNA regulating salt stress through relevant signaling pathways was proposed,especially the regulation of jasmonate signaling pathway significantly regulating target genes under the influence of salt and endophytic fungi.The co-expression analysis of the differential miRNAs and mRNA was done in this study.4 pairs of coanalysis results were obtained.The expression of hvu-miR156 in E+200 vs E+CK was up-regulated,and its target gene MLOC₁3032 was down-regulated in the transcriptome.Then the real-time fluorescence quantification results verified this result again.