Screening Of Genes Related To Biosynthesis Of Herbicidal Active Metabolites From Epicoccum Sorghinum

The research group previously isolated a pathogenic fungus,called Epicoccum sorghinum,from the leaves of Digitaria sanguinalis,which belongs to the phylum Ascomycota,Pleosporales,Epicoccum.The pathogenic fungus can quickly infect and kill D.sanguinalis seedlings.Its main active secondary metabolite,Epoxydon,has good herbicidal activity and has the potential to be developed as a bioherbicide.To clarify its biosynthetic pathway is the basics for its further research and application.In order to screen the genes involved in Epoxydon biosynthesis,this study used high-throughput sequencing technology to perform whole genome sequencing of E.sorghinum.Transcriptome sequencing was performed on the infected host and conventional culture E.sorghinum to further screen the predicted gene clusters.The q RT-PCR reference genes of E.sorghinum under different treatment conditions were screened,and the transcriptomic sequencing results were verified.1.The whole genome of E.sorghinum was sequenced and assembled,the genome size was32957594 bp,22 contigs were obtained,11052 coding genes were predicted,and the coding regions accounted for 50.53% of the whole genome.The E.sorghinum secondary metabolite gene clusters were predicted by the anti SMASH program,and a total of 30 secondary metabolite gene clusters were obtained,including 350 genes.The contig13.1 gene cluster was similar to patulin biosynthesis gene cluster.Epoxydon was an intermediate product of patulin biosynthesis pathway.The 6 genes in contig13.1 gene cluster A01551,A01552,A01553,A01554,A01556 and A01558 were related to the gene before Epoxydon in patulin biosynthesis pathway respectively Pat K 6-methylsalicylic acid synthase,Pat J gene hypothetical protein,Pat I m-hydroxybenzyl alcohol hydroxylase,Pat G amidohydrolase,Pat H m-cresol methyl hydroxylase and Pat M ABC transporter.The similarity of them were 50-80%.Therefore,contig13.1 gene cluster was closely related to the biosynthesis of Epoxydon in E.sorghinum.2.Transcriptome sequencing of infected D.sanguinalis leaves and conventionally cultured E.sorghinum strains showed a total of 686 genes with significant differences in expression,489 up-regulated genes and 197 down-regulated genes.Among them,there are 35 genes related to the biosynthesis of secondary metabolites,27 genes up-regulated and 8 genes down-regulated;62 differential genes related to fungal pathogenicity,47 genes up-regulated in expression and 15 genes down-regulated.The genes A02639,A02957,A02961,A02962,and A02963,which are related to the biosynthesis of secondary metabolites and their expression levels were significantly up-regulated,all belong to the contig16.2 gene cluster,the results suggested that secondary metabolites related to contig16.2 gene cluster may play an important role in the host infection process of E.sorghinum.The expression levels of A01551,A01552,A01553,A01555,A01556,and A01558 in the contig13.1 gene cluster did not change significantly during the infection of E.sorghinum,but these genes all had relatively high expression levels.These results indicated that the synthetic products related to the gene cluster had important biological functions.Epoxydon is the main herbicidal active component in the secondary metabolites of E.sorghinum,which may be related to the high expression of contig13.1 gene cluster.3.The stable q RT-PCR reference genes of E.sorghinum under different treatment conditions were screened.The most stable reference gene for E.sorghinum in the process of infecting the host was TBP,and the most stable reference gene under different culture time conditions was His-H3-1.Five genes were selected from the contig13.1 gene cluster,contig16.2 gene cluster and other differentially expressed genes,and the transcriptome results were verified by q RT-PCR.The results showed that four genes in the contig13.1 gene cluster had higher the relative expression levels of genes;the differential expression of 5 genes in the contig16.2 gene cluster and 4 genes in other differentially expressed genes was consistent with the transcriptome analysis.