Analysis Of The Differenially Expressed Genes About Swainsonine Of Undifilum Oxytropis By RNA-seq

Locoweeds are the poisonous species of the genera Astragalus and Oxytropis(Fabaceae),the toxic plants have resulted in valuable economic losses to animal husbandry annually.The primary reason for animal poisoning caused by locoweeds is the bioactive substance(Swainsonine,SW)within locoweeds.The toxin in the locoweed is not produced directly by the plant but by endophytic fungi.Many studies have shown that there is a swainsonine-producing fungal endophyte in locoweeds widespreadly,and there is a positive correlation between swainsonine concentration and the presence of Undifilum oxytropis in locoweeds.In our lab,the fungal endophytes were isolated and identified which were from locoweeds in China,and also analysised genetic polymorphisms.Effects of putative precursors were invested by feeding on the production of swainsonine by the endophytic Undifilum oxytropis fungi.The study on the proteomic of locoweed fungal endophytes synthesizing swainsonine were to identify the enzyme related to swainsonine biosynthsis pathway.The objective of this study is that RNA sequencing technology was used to transcriptome sequencing of two strains of Undifilum oxytropis of locoweeds.Both of them can produce indolizidine alkaloids swainsonine,but the swainsonine concentration of FEL4-F5 is nearly fifty-seven times higher than that of FEL1-A5.Combineded with bio-technology platform,the differentially expressed genes were selected which were related to swainsonine concentration.It provides important theoretical basis for the further research of swainsonine biosynthsis pathway in Undifilum oxytropis endophytic fungi of locoweeds.The main results were as follows:1.Using RNA-seq technology,39 and 49 million clean reads were obtained from FEL1a-A5 and FEL4-F5,respectively.It assembled 12,312 Unigenes by De novo.There were 10,393 Unigenes mapped to Nr?Swiss-Prot?KEGG and KOG database.It provides reference sequences for analysis of the genes related to swainsonine concentration.2.In total,2,663 genes were identified as differentially expressed genes.Among them,1,651 genes expressed up-regulatedly and 1,012 genes expressed down-regulatedly.There were 94 metabolic pathways including 2,224 differentially expressed genes which were found by KEGG.Most of differentially expressed genes were categorized into catalytic activity and oxidoreductase activity terms by GO-term analysis.3.Among the differentially expressed genes,genes of 5 enzymes,which were related to swainsonine biosynthsis pathway in Undifilum oxytropis,were identified by bioinformatics,including 1 saccharopine dehydrogenase,1 L-pipecolate oxidase,3 pyrroline-5-carboxylate reductase,16 polyketide synthase and 21 cytochrome P450.4.11 differentially expressed genes were selected and quantitive real-time PCR were further performed to confirm their expression patterns during two strains of fungal endophytes FEL4-F5 and FEL1-A5.The results showed that following,1 saccharopine dehydrogenase gene,1 polyketide synthase gene and 1 pyrroline-5-carboxylate reductase gene were obviously decreased,while 1 L-pipecolate oxidase gene,3 pyrroline-5-carboxylate reductase genes,1 polyketide synthase gene,and 3 P450 genes were increased.which is consistent with the results of RNA-Seq.The results indicate that the differentially expressed genes obtained by RNA-Seq is reliable.