| In this study,we utilized an endophytic fungi strain 1-G4 isolated from the epidermal tissue of wild Bletilla striata tuber,which showed pro-growth effect,as experimental material.The genome of 1-G4 was sequenced;the effects of symbiosis between the strain and B.striata seedling on the agronomic traits,physiological and biochemical response and drought resistance characteristics of host plant B.striata were studied;and the molecular mechanism of 1-G4promoting B.striata growth was discussed from the perspective of transcriptomics and metabolomics.The results were as follows:1.1-G4 was identified as Aspergillus flavus.Observation under the microscope showed that 1-G4 showed the characteristics of Aspergillus.The phylogenetic tree showed that 1-G4 and Aspergillus flavus clustered in the same branch with 87%self-spreading rate.2.The 1-G4 genome data was obtained.Genes related to the synthesis of plant hormones and phenols were screened.28 conting sequences were finally assembled.The genome size was 37484600 bp,N50 was 2498349 bp,and the overall integrity was 98.3%;gene function was annotated:a total number of11774 gene sequences were annotated,of which the annotation rate of both NR and Tr EMBL database was over than 99%;949 secretory proteins were identified,and many genes related to the synthesis of hormones,polysaccharides and phenols were found in B.striata.8,14 and 29 genes involved in the metabolic pathway of starch and sucrose,mutual conversion of pentose and glucuronic acid,and metabolic pathway of galactose respectively were identified.3.1-G4 elicitor and spore suspensions could promote the growth of B.striata seedlings.The morphological indexes of B.striata seedling could be promoted by 1-G4 elicitors and spore suspensionsthe:the morphological indexes of B.striata treated with 1-G4 elicitors and spore suspensions were determined.The pro-growth was found from the results of the experiments of B.striata treated with 1-G4 elicitor and spore suspensions:10%1-G4 elicitor could significantly promote the height and fresh weight of B.striata seedling;the symbiosis of 2.5,5.0,7.5 and 10.0 m L(10~51-G4 spores per m L)1-G4 with B.striata seedling could increase leaf length and leaf area of B.striata seedling and promote the root growth,in which the concentration of 10.0 m L 1-G4 spore suspension had the most obvious effect.4.1-G4 spore suspensions could increase the activity of the stress resistance related enzymes and the content of medicinal components of B.striata seedling.The physiological and biochemical characteristics of B.striata symbiotic seedlings were determined.The results showed that 1-G4 spore suspensions could improve the activities of CAT in B.striata symbiotic seedlings.10.0 m L treatment group was the most effective:the activities of 14 d,18 d and 20 d were 166.67%,468.42%and 124.36%of the control group respectively;1-G4 can reduce the MDA content,among which,the MDA content of 10 m L treatment group was 54.18%,89.83%,56.80%and 73.29%of the control group at 12 d,14 d,16 d and 20 d,respectively,which was the most effective for reducing MDA content;1-G4 can increase the contents of total sugar and total phenol in B.striata seedling.In 10.0 m L treatment group,the total sugar content on 16 and 18 days were 292.09%and 136.61%of the control group,and the total phenol content on 16 and 20 days were 112.07%and180.49%of the control group,respectively.These results showed that 1-G4could maintain a normol growth condition for B.striata seedling by increasing the activity of antioxidant enzyme and decreasing the content of MDA,as well as increasing the content of total sugar and total phenol.Among them,10 m L was the optimal concentration of 1-G4 spore suspensions.5.1-G4 spore suspensions could increase the ability of drought stress resistance of B.striata seedling.Physiological and biochemical characteristics of 1-G4-B.striata symbiotic seedlings under drought stress were measured.The results showed that 2.5 m L 1-G4 spore suspension showed the best drought resistance under 5%,10%,15%and 20%PEG stress conditions:the maximum effect of OD,CAT,MDA,PRO and soluble protein was 156.60%,381.81%,41.18%,263.5%and 169.00%of the control,respectively.The results showed that 1-G4 spore suspensions could enhance the activities of SOD and CAT,increase the contents of PRO and soluble protein,and decrease the content of MDA,thus enhancing the ability of B.striata seedling to resist drought stress.Among them,2.5m L 1-G4 spore suspension had the best promotion effect.6.The transcriptome data of B.striata seedling treated with 1-G4 elicitor was obtained.The transcriptome and analysis were performed on the B.striata seedlings treated with 1-G4 elicitor.The results of transcriptome analysis of B.striata treated with 1-G4 elicitor showed that,a total of 84.32GB clean data was obtained.The clean data of each sample was more than 6.8GB,and the percentage of Q30 bases was more than 93.91%.The GC content ranged from46.13%to 46.93%.A total of 193003 transcripts were obtained by splicing transcriptome sequences,with an average length of 1462 bp,the length of N50of 2461 bp and the total number of nucleotides of 282085198 bp.95657unigenes were obtained,with the average length of 1155 bp,the maximum length of 24353 bp,the length of N50 of 919 bp,and the total number of nucleotides of 110450720 bp.These results showed that the sequencing assembly integrity was high.The highest proportion of Unigene annotated to NR database is 43.41%,and the proportion annotated to GO,KO and Swissprot databases are25.48%,9.58%and 24.62%respectively.The differential genes between different treatment groups were screened by using FDR less than 0.01 and difference multiple FC(fold change)greater than or equal to 2.In the comparison group of control vs 5%1-G4,a total of 1657 differential genes were screened,of which 763 differential genes were up-regulated and 894 differential genes were down regulated.In the comparison group of control vs 10%1-G4,2415 differential genes were identified,including 1208 up-regulated differential genes and 1207 down regulated differential genes.7.1-G4 elicitor can promote the growth of B.striata seedling by regulating hormone levels.Metabolome analysis was performed on the B.striata seedlings treated with 1-G4 elicitor:11 hormones were found,including ME-IAA,IP,TZ,DZ,H2JA,ABA,ACC,GA7,GA15,GA19 and GA20.With the increase of1-G4 elicitor concentration,the contents of SA,ICAld and ME-IAA were significantly increased while the contents of IP and ACC were significantly decreased.8.1-G4 could regulate the expression of the hormone-related genes of B.striata seedling,thereby regulating the hormone level of it,and ultimately promoting the growth of B.striata seedling.The results showed that 11hormones were related to 139 genes,and ME-IAA,IP and ACC were closely related to the concentration of 1-G4 elicitor.Among them,22 genes were involved in ME-IAA,38 genes were involved in IP,and 2 genes were involved in ACC. |
