| Amanita spp,one of macro-fungi attracted much attention,not only contains some famous edible fungi,but also includes many deadly virulent fungi.In the poisoning cases of poisonous mushrooms,liver injury type highly toxic Amanita species causing are the crucial culprits leading to the death of patients,and its main lethal toxin is cyclic peptide toxin.Studies have shown that cyclopeptide toxins were encoded by MSDIN gene family,and the key enzyme of its biosynthesis was prolyl oligopeptidase(POP).It is now known that some lethal Amanita species with acute liver failure contain rich MSDIN genes and have the extensive ability of cyclic peptide synthesis.However,whether those edible neurotoxicity,acute renal failure toxicity and unknown toxicity Amanita species also have MSDIN and POP genes or not? Can they synthesize similar cyclic peptides? What is the difference of the cyclic peptide and POP genes between highly toxic and non-highly toxic Amanita species?These scientific questions need further systematically study.Therefore,the transcriptomes of15 Amanita species with different toxicity were sequenced,and the diversity of cyclic peptide genes and the key enzyme gene POP of its biosynthesis were analyzed.The results were as follows:1.Transcriptome sequencing analysis: For the first time,BGISEQ-500 and DNBSEQ sequencing platforms were used for the de novo transcriptome sequencing of the fruiting bodies from 15 Amanita species(including edible Amanita,highly toxic Amanita with liver damage,toxic Amanita with acute renal failure,neuropsychiatric Amanita and the species of unknown toxicity).And,high-quality transcriptome with N50 length of 2 532-5 359 bp were obtained.The sequencing results showed that the number of unigenes obtained from 15 Amanita species ranged from 28 104 to 66 338.The edible A.ochracea and A.sinensis had the most unigenes and more than 60 000 in number,and were more twice than that of A.oberwinklara possessing the least unigenes.Seven databases were used for function annotation of unigenes,and the largest number of unigenes,71.30%-94.19% of all the unigenes,were annotated in NR database.2.Research on cyclic peptide gene family: Local blast software was used to retrieve the cyclic peptide coding gene MSDIN from the obtained transcriptome data.The specific primers were designed using the retrieved relevant gene sequences,and were used for PCR amplification and sequencing verification.The results showed that the sequences of 75 cyclic peptide coding gene family were obtained from 15 species of Amanita spp.,and 55 cyclic peptides composed of 6-10 amino acids were predicted,of which 26 new cyclic peptides were not reported.Cluster analysis of 75 cyclic peptide precursor peptide sequences in this study and 276 reported sequences showed that all precursor peptides generated eight branches,namely Amanita peptides,phallotoxin peptides,cycloamanide F,cycloamanide,amanexitide,and unknown peptides I,II and III.According to the phylogenetic tree,it was speculated that cyclic peptides with similar functions can be clustered.We preliminarily estimated that 7 unknown cyclic peptides should be new toxin compounds of phallotoxin peptides and 11 unknown cyclic peptides should be new non toxin cyclic peptide compounds.3.Research on POP gene,the key enzyme of cyclic peptide biosynthesis: POP gene was retrieved from the obtained transcriptome data using local blast software.Specific primers were designed on the basis of the retrieved relevant gene sequences,and were used for PCR amplification and sequencing analysis.15 POPA genes and 5 POPB genes were obtained from 15 species of Amanita.The full length of POPA genes varied from 3 173 to 3239 bp,most of which were composed of 18 introns and 19 exons,and encoded 757 to 763 amino acids.The full length of POPB gene varied from 3 025 to 3 096 bp,most of which were composed of 18 introns and 19 exons,and encoded 727-731 amino acids.Phylogenetic analysis of 20 POP sequences obtained in this study and 79 reported sequences showed that the POPA of all Amanita species were clustered into one clade,and the POPB of Amanita and other highly toxic species were clustered into another clade.In this study,the transcriptome data of 15 Amanita species were obtained,of which 13 were reported for the first time,laying important foundation for the functional gene mining and related research in Amanita.This study showed that the highly toxic Amanita containing toxic cyclic peptides had rich diversity of cyclic peptide coding genes and synthesis ability of extensive cyclic peptides(including toxic cyclic peptides or non toxic cyclic peptides).Non highly toxic Amanita had no or less cyclic peptide coding genes,and the cyclic peptide synthesis ability was very limited.In this study,POPB gene was found in non highly toxic Amanita for the first time,breaking the view that POPB gene only existed in highly toxic Amanita species containing Amanita cyclic peptide.The new unknown cyclic peptides predicted in this study will provide scientific basis for their isolation and identification,activity and function study. |
