Molecular Cloning Of Genes Involved In Ophiobolin A Biosynthesis Pathway From Mycoherbicidal Fungus

Ophiobolins are sesterterpenoid-type phytotoxins produced by fungi. They are active on a broadspectrum of organisms including plants, fungi, bacteria, nematodes and tumor cells, so they maybe important candidate for development of new crop-protection and pharmaceutical product.However, due to the low content of bioactive components in ophiobolin-producing fungi, thecommercial applications of ophiobolins are restricted. Hence, a number of studies have beenconducted in attempts to improve the yield of ophiobolins.To significantly increase yields of ophiobolin A from an ophiobolin-producing fungus,Biopolaris eleusines (Be), this study focused on two aspects. The first was that transcriptomesequencing was used to produce a substantial expressed sequence tags (EST) dataset from Be.Based on the analysis of EST, molecular cloning by RACE technology and analysis of related keygenes involved in ophiobolin A biosynthesis pathway were conducted. This would provide someimportant candidate genes for Be genetic engineering to improve the yield of ophiobolin A in thefuture. The second was the stimulatory effect of methyl jasmonate (MeJA) on the expression levelof BeHMGR, BeIPPI, BeFPPS, BeGGPPS involved in biosynthesis of ophiobolin A in Be. Themain results from the study were as follows:To investigate the profile of gene expression in Be and elucidate its functional gene,high-throughput transcriptome sequencing was firstly used to produce a substantial EST from Be.A total of26,555,560high quality EST with an average read length of559bp were generated.These EST were assembled into32,100unigenes.97.9%of these unigenes (31432) wereannotated and showed different levels of homology with other organisms using BLAST searches(E-value≤1e-5) against the SwissProt, KEGG, Nr, COG and GO databases. Sixty unigenes(encoding17enzymes) were found to be involved in terpenoid biosynthesis. Seventy-six unigeneswere also analyzed be similar to that of transcription factor genes. Data presented in this study notonly provided a number of resources in the profile of gene expression of Be, but also laied thefoundation for cloning and analyzing the encoding gene and regulation gene involved inophiobolins biosynthesis.A full-length cDNA encoding3-hydroxy-3-methylglutaryl-CoA reductase gene (designated asBeHMGR, GenBank accession No. JQ780844) was cloned from the ophiobolin A-producingfungus Be by rapid amplification of cDNA ends (RACE) technique and was subject to detailedbioinformatic analysis. The full-length cDNA of BeHMGR was3906bp containing a3474bpORF encoding1157amino acids. BLASTP analysis revealed that the deduced BeHMGR hadextensive homology with HMGR of other fungi. Phylogenetic tree analysis indicated thatBeHMGR belongs to the fungi HMGR super-family and had the closest relationship with HMGRfrom Pyrenophora tritici-repentis (86%identity and93%similarity). The expression of BeHMGRwas up-regulated by methyl jasmonate (MeJA), suggesting that it might be elicitor-responsive.A full-length cDNA encoding isopentenyl diphosphate isomerase gene (designated as BeIPPI, GenBank accession No. JQ780840) was isolated and cloned from the ophiobolin A-producingfungus Be by RACE technique. The bioinformatic analysis found that the full-length cDNA ofBeIPPI was1059bp containing a672bp ORF encoding a polypeptide of223amino acids.Analysis of BeIPPI genomic DNA revealed that it contained two exons and one intron. BLASTPanalysis revealed that the deduced BeIPPI contained all conserved domains and completelyidentical amino acids of catalytic active site compared to other fungi IPPI, but about40aminoacids of BeIPPI was abridged at C-terminus. Functional analysis of BeIPPI will be required toverify that if it is a functional protein.A full-length cDNA encoding farnesyl diphosphate synthase gene (designated as BeFPPS,GenBank accession No. JQ780841) was obtained from the ophiobolin A-producing fungus Be byRACE and was subject to detailed bioinformatic analysis. The full-length cDNA of BeFPPS was1186bp containing a1044bp ORF encoding a polypeptide of347amino acids. Analysis ofBeFPPS genomic DNA revealed that it contained two exons and one intron. BLASTP analysisrevealed that the deduced BeFPPS had extensive homology with other fungi FPPS. Phylogenetictree analysis indicated that BeFPPS belongs to the fungi GGPPS super-family and had the closestrelationship with FPPS from P. teres f. teres(89%identity).A full-length cDNA encoding geranylgeranlyl diphosphate synthase gene (designated asBeGGPPS, GenBank accession No. JQ780842) was cloned from Be by RACE. The bioinformaticanalysis indicated that the full-length cDNA of BeGGPPS was1666bp containing a1305bp ORFencoding a polypeptide of434amino acids. Analysis of BeGGPPS genomic DNA revealed that itcontained three exons and two introns. BLASTP analysis revealed that the deduced BeGGPPS hadextensive homology with other fungi GGPPS contained all five conserved domains and functionalaspartate-rich motifs of the prenyltransferases. Phylogenetic tree analysis indicated that BeGGPPSbelongs to the fungi GGPPS super-family and had the closest relationship with GGPPS from P.tritici-repentis.The stimulatory effect of MeJA on the transcriptional level of ophiobolin A gene in Be was alsopreliminarily studied. QRT-PCR analysis indicated that gene expression of BeHMGR, BeIPPI,BeFPPS and BeGGPPS, four key enzymes in ophiobolin A biosynthesis pathway, wasdramatically raised by MeJA treatment.Transcriptome sequences of Be was firstly determined. BeHMGR, BeIPPI, BeFPPS andBeGGPPS of four key enzymes involved in ophiobolin A biosynthesis pathway were isolated fromophiobolin A-producing fungus stratin Be for the first time. Furthermore, the induction of MeJAon expression level of BeHMGR, BeIPPI, BeFPPS and BeGGPPS was preliminarily studied.These information might be helpful not only for theoretical research of further defining themechanism of ophiobolin A biosynthesis, but also supply more clues of target enzymes tomodulate the production of ophiobolin A for practical application.